Sordarin derivatives

ABSTRACT

Sordarin derivatives are antifungal agents useful in the treatment and/or prevention of human and animal fungal infections, as well as in the control of phytopathogenic fungi in crops.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is based on, and claims priority from, provisionalapplication No. 60/026,993 filed on Oct. 7, 1996 which is herebyincorporated by reference in its entirety.

SUMMARY OF THE INVENTION

The present invention relates to sordarin derivatives which are potentantifungal agents with a broad spectrum of activity, to processes fortheir preparation, to pharmaceutical and agricultural compositionscontaining the compounds, and to methods of controlling fungalinfections in human, animals and plant materials using such compounds.

BACKGROUND OF THE INVENTION

Sordarin is an antifungal antibiotic isolated from the mould Sordariaaraneosa (see GB 1,162,027 and Helvetica Chimica Acta, 1971, 51:119-20).Other compounds having the sordarin skeleton have also been reported asantifungal agents. Japanese Kokai J62040292 discloses the compoundzofimarin isolated from Zofiela marina sp.; Japanese Kokai J06157582discloses the compound BE-31405 isolated from Penicillium sp.; andSCH57404 is reported in J. Antibiotics, 1995, 48:1171-1172.Semi-synthetic sordarin derivatives are reported in PCT ApplicationsWO96/14326 and WO96/14327.

Sordaricin, the aglycon, may be obtained from sordarin by acidhydrolysis (Hauser and Sigg, Helvetica Chimica Acta, 1971, 51:119-20);similarly sordaricin methyl ester is obtained from sordarin methylester. The total synthesis of sordaricin methyl ester is reported inKato et al, J. Chem. Soc. Chem. Commun., 1993, 1002-1004, which alsodiscloses O-methoxymethyl sordaricin methyl ester. The diacetate of4-desformyl-4-hydroxymethyl sordaricin is disclosed in Mander andRobinson, J. Org. Chem., 1991, 56(11):3395-3601. Neither sordaricin northe reported derivatives thereof has been shown to have biologicalactivity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compounds having the formula I: ##STR1##wherein R is

(a) C(═O)OR¹,

(b) C(═O)NR² R³,

(c) C(═O)R⁴,

(d) CH(R²)OR⁵,

(e) C(R⁶)(R⁷)(R⁸), ##STR2## with the proviso that when R¹² is CHO, R isnot (g); R¹ is

(a) C₁ -C₁₄ alkyl,

(b) C₂ -C₁₄ alkenyl,

(c) C₂ -C₁₄ alkynyl,

(d) C₃ -C₂₀ cycloalkyl,

(e) C₃ -C₂₀ cycloalkenyl,

(f) aryl or

(g) aryl C₁₋₆ alkyl;

R² and R³ are independently

(a) H or

(b) R¹ ;

R⁴ is

(a) H,

(b) R¹ or

(c) --(CH₂)_(m) NR² R³ ;

R⁵ is

(a) R¹ or

(b) --(CH₂)_(x) O(CH₂)yH;

R⁶ is

(a) H,

(b) R¹,

(c) --(CH₂)_(y) CHR₁₁ (CH₂)_(z) H,

(d) --(CH₂)_(y) C.tbd.C(CH₂)_(z) H,

(e) --(CH₂)_(y) C(R⁷)═CH(CH₂)_(z) H,

(f) --(CH₂)_(y) C.tbd.C(CH₂)_(m) R¹¹,

(g) --(CH₂)_(y) C(R⁷)═CH(CH₂)_(m) R¹¹,

R⁷ and R⁸ are independently

(a) H, or

(b) C₁ -C₁₄ alkyl;

R⁹ and R¹⁰ are independently

(a) H,

(a) C₁ -C₁₄ alkyl,

(a) C₂ -C₁₄ alkenyl,

(a) aryl C₁₋₆ alkyl;

R¹¹ is

(a) OR⁷,

(b) OCH₂ CH₂ (CH₂)_(n) OR⁷, or

(c) NR² R³ ;

R¹² is

(a) --C(═O)R¹⁴,

(b) --CH═NOH, or

(c) --CH₂ OCH₃ ;

R¹³ is

(a) H,

(b) --CH₂ C₆ H₅,

(c) --CH₂ CH═CH₂, ##STR3## R¹⁴ is (a) H,

(b) C₁ -C₄ alkyl,

(c) --CCl₃,

(d) --CBr₃,

(e) --CF₃, or

(f) OH;

n is 0 or 1;

m is 1-6;

x is 2-6;

y is 0-6;

z is 0-6 or

a pharmaceutically or agriculturally acceptable salt thereof.

One embodiment of the present invention provides compounds of formula Iwherein

R¹² is --CHO;

R is

(a) C(═O)OR¹,

(b) C(═O)NR² R³,

(c) C(═O)R⁴,

(d) CH₂ OR⁵,

(e) C(R⁶)(R⁷)(R⁸), ##STR4##

Another embodiment of the present invention provides compounds offormula I wherein

R¹² is --CHO

R¹ 3 is H,

R is C(═O)OR¹.

Another embodiment of the present invention provides compounds offormula I wherein

R¹² is --CHO

R¹³ is H,

R is C(═O)NR² R³.

Another embodiment of the present invention provides compounds offormula I wherein

R¹² is --CHO

R¹³ is H,

R is C(═O)R⁴.

Another embodiment of the present invention provides compounds offormula I wherein

R¹² is --CHO

R¹³ is H,

R is C(R²)OR⁵.

Another embodiment of the present invention provides compounds offormula I wherein

R¹² is --CHO

R¹³ is H,

R is C(R⁶)(R⁷)(R⁸).

Another embodiment of the present invention provides compounds offormula I wherein

R¹² is --CHO

R¹³ is H;

R is ##STR5##

A preferred embodiment of the present invention provides compounds offormula I wherein

R¹² is --CHO,

R¹³ is H,

R is CH(R⁶)(R⁷),

R⁶ is

(a) H,

(b) R¹

(c) --(CH₂)_(y) CHR¹¹ (CH₂)_(z) H,

(d) --(CH₂)_(y) C(R⁷)═CH(CH₂)_(z) H,

R⁷ is H or C₁ -C₆ alkyl,

R¹¹ is OR⁷.

Another preferred embodiment of the present invention provides compoundsof formula I wherein

R¹² is --CHO

R¹³ is H,

R is

(a) --CH₃,

(b) --CH₂ CH₃,

(c) --CH₂ CH₂ CH₃,

(d) --CH₂ CH₂ CH₂ CH₃,

(e) --CH₂ CH₂ CH₂ CH₂ CH₃,

(f) --CH₂ CH₂ CH₂ CH₂ CH₂ CH₃,

(g) --CH₂ CH₂ CH₂ CH₂ CH₂ CH₂ CH₃,

(h) --CH(CH₃)₂,

(i) --CH₂ CH(CH₃)₂,

(j) --CH₂ CH₂ CH(CH₃)₂,

(k) --CH₂ C₆ H₅,

(l) --CH₂ CH═CHCH₃,

(m) --CH₂ CH═CHCH₂ CH₃,

(n) --CH₂ CH═CHCH₂ CH₂ CH₃,

(o) --CH₂ CH═CHCH₂ CH₂ CH₂ CH₃

A more preferred embodiment of the present invention provides compoundsof formula I wherein

R¹² is --CHO

R¹³ is

(a) H,

R is

(a) --CH₂ CH₂ CH₃,

(b) --CH₂ CH₂ CH₂ CH₃,

(c) --CH₂ CH₂ CH₂ CH₂ CH₃,

(d) --CH₂ CH(CH₃)₂,

(e) --CH₂ CH₂ CH(CH₃)₂,

(f) (Z)--CH₂ CH═CHCH₂ CH₃,

(g) (Z)--CH₂ CH═CHCH₂ CH₂ CH₃.

In another aspect of the present invention, there is provided apharmaceutical composition which comprises an antifungal effectiveamount of a compound of formula I, and a pharmaceutically acceptablecarrier. Also provided is a pharmaceutical composition which is made bycombining a compound of formula I and a pharmaceutically acceptablecarrier.

Another aspect of the present invention provides an agriculturalcomposition which comprises an antifungal effective amount of a compoundof formula I, and an agriculturally acceptable carrier thereof. Alsoprovided is an agricultural composition which is made by combining acompound of formula I and an agriculturally acceptable carrier.

Yet another aspect of the present invention provides a method fortreating fungal infection in an animal (including humans) whichcomprises administering to an animal in need of such treatment anantifungal effective amount of a compound of formula I.

A further aspect of the present invention provides a method forcontrolling phytopathogenic fungi in plants which comprises applying tosaid plant an antifungal effective amount of a compound of formula I.

As used herein, unless otherwise specified, the following terms have theindicated meanings.

The term "alkyl", alone or as part of a group (e.g. aralkyl), means astraight or branched chain alkyl moiety, optionally substituted withcycloalkyl or cycloalkenyl, having the designated number of carbon atomssuch as methyl, ethyl, n-propyl, n-butyl, isopropyl, isobutyl,isopentyl, s-butyl, t-butyl, n-hexyl, n-octyl, decyl, undecyl,cyclopropylmethyl, cyclobutylmethyl, 2-cyclopentylethyl,cyclododecylmethyl, cyclohexylmethyl and the like.

The term "cycloalkyl" means a saturated carbocycle containing one ormore rings of from 3 to 12 carbon atoms, optionally substituted withC₁₋₃ alkyl. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl,cyclopentyl, cyclohexyl, methylcyclohexyl, cycloheptyl, adamantyl,2-ethyl-1-bicyclo[4.4.0]decyl, and the like.

The term "aryl", alone or as part of a group (e.g. aralkyl), includesphenyl, biphenyl, terphenyl, naphthyl, anthracenyl or heteroaryl eachoptionally substituted by one to three groups independently selectedfrom halogen, hydroxyl, carboxyl, C₁₋₆ alkyl, C₁₋₆ alkoxy or C₁₋₄alkoxycarbonyl. The heteroaryl group may be a 5- or 6-memberedheteroaromatic ring containing one or more heteroatoms selected fromnitrogen, oxygen and sulfur. Suitable examples of heteroaryl groupsinclude pyridyl, quinolinyl, furyl, thienyl and pyrrolyl.

The term "alkenyl" means a straight or branched carbon chain having atleast one carbon-carbon double bond. Examples include vinyl, allyl,butenyl, isobutenyl, butadienyl, and the like.

The term "cycloalkenyl" means an unsaturated carbocycle containing oneor more rings of from 3 to 12 carbon atoms, optionally substituted withC₁₋₃ alkyl. Examples of cycloalkyl groups are cyclobutenyl,cyclopentenyl, cyclohexenyl, methylcyclohexenyl, and the like.

The term "alkynyl" means a straight or branched carbon chain having atleast one carbon-carbon triple bond. Examples include acetylenyl,propargyl, butynyl, 1,3-pentadiynyl, and the like.

The term "controlling", used in association with phytopathogenic fungi,includes prophylactic use (i.e. to protect against infection) andcurative use (i.e. to eradicate infection).

The term "plants" include live plants, foliage, flowers, seeds, fruits,and other materials derived from plants. The term also includes roots ofthe plant via application of the active ingredient to the soil.

The term "composition", as in pharmaceutical or agriculturalcomposition, is intended to encompass a product comprising the activeingredient(s), and the inert ingredient(s) that make up the carrier, aswell as any product which results, directly or indirectly, fromcombination, complexation, aggregation, or other interactions of any twoor more of the ingredients, or from dissociation of one or more of theingredients, or from other types of reactions of one or more of theingredients.

Suitable salts of a compound of formula I include inorganic base saltssuch as alkali metal salt (e.g. sodium and potassium salts), ammoniumsalts, and organic basesalts. Suitable organic base salts include aminesalts such as tetraalkylammonium (e.g. tetrabutylammonium ortrimethylcetylammonium), trialkylamine (e.g. triethylamine),dialkylamine salts (e.g. dicyclohexylamine), optionally substitutedbenzylamine (e.g. phenylbenzylamine or p-bromobenzylamine),ethanolamine, diethanolamine, N-methylglucosamine, N-methylpiperidine,pyridine and substituted pyridine (e.g. collidine, lutidine,4-dimethylaminopyridine), and tri(hydroxymethyl)methylamine salts, andamino acid salts (e.g. lysine or arginine salts).

Compounds of formula I are prepared from sordarin (II) or its aglycone,sodaricin. Sordarin is[1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a[(6-deoxy-4-O-methyl-β-D-altropyranosyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid having the formula II: ##STR6##

Sordarin can be obtained by the cultivation of Sordaria araneosa NRRL3196 (also deposited with the ATCC as ATCC 36386) according to theprocedure described in GB1,162,027 or in WO96/14326. Sordarin can alsobe isolated from the fermentation of Rosellinia subiculata (ATCC 74386)or an unidentified fungus (ATCC 74387) as described hereinbelow. Bothcultures were deposited on Aug, 27, 1996 in the permanent collection atthe American Type Culture Collection, 12301 Parklawn Drive, Rockville,Md, 20852, USA under the terms of The Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure.

Sordaricin (III) is[1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]4-formyl-8a-(hydroxymethyl)-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid having the formula III: ##STR7## Sordaricin can be prepared fromsordarin by treatment with concentrated hydrochloric acid. As disclosedin WO96/14326 sordaricin is also obtained from fermentation of a mutantderived from Sordaria araneosa NRRL 3196, and by biotransformation ofsordarin using a Coryneform species.

The compounds of the present invention (formula I) may be prepared bythe processes described below. The conditions are representative and arenot meant to be limiting.

Scheme 1 shows the conversion of sordarin or a derivative of sordarin toits 4-aldoxime or 4-methoxymethyl derivative. The aldoxime may beprepared by treating the aldehyde compound with hydroxylaminehydrochloride in an alcohol-pyridine solvent system. The methoxymethylcompound may be prepared by first protecting the carboxylic acid groupof sordarin or a derivative (such as a benzyl ester for example),followed by reduction of the aldehyde with sodium borohydride in analcohol solvent. The resultant alcohol is methylated in the presence ofa strong base such as sodium hydride with methyl iodide. Removal of theprotecting group produces a compound of formula I. ##STR8##

Scheme 2 depicts the synthesis of carbamate, ester and carbonatederivatives of sordarin aglycone. The preparation of carbamates may becarried out by treatment of suitably protected sordarin aglycone with anisocyanate (in the examples where R³ is H) or a carbamoyl halide orother activated carbamoylating agent in an inert solvent. Removal of theprotecting group produces a carbamate compound of formula I.

Ester derivatives may be prepared in a similar fashion by treatment ofprotected sordarin aglycone with an activated carbonyl compound such asan acid chloride or mixed anhydride preferably in the presence of anacylation catalyst such as N,N-dimethylaminopyridine and a base such aspyridine. Removal of the protecting proup yields an ester compound offormula I.

Carbonate derivatives may be prepared by the treatment of protectedsordarin aglycone with an activated carbonate such as a chloroformate orpyrocarbonate. An acylation catalyst such as N,N-dimethylaminopyridineand a base such as pyridine is preferably employed in the reactionmixture. Removal of the protecting proup yields a carbonate compound ofFormula 1. ##STR9##

Scheme 3 shows the synthesis of ether derivatives of sordarin aglycone.Treatment of the carboxylic acid-protected aglycone with an α-haloetherunder basic conditions or a vinyl ether under acidic conditions givesrise to the substituted α-alkoxyether derivatives. Treatment ofprotected sordarin aglycone with a primary or secondary halide orsulfonate and a suitable base such as sodium hydride under S_(N) 2conditions gives the corresponding primary or secondary etherderivatives whereas treatment of the aglycone with a tertiary alcohol,halide or sulfonate and a Lewis acid under S_(N) 1 conditions gives thecorresponding tertiary ether derivative. Removal of the protecting groupfrom the compound gives a compound of formula I. ##STR10##

The preparation of cyclic acetals from protected sordarin aglycone isdepicted in Scheme 4. Treatment of the aglycone with a cyclic vinylether in the presence of an acid catalyst followed by removal of theprotecting group gives a compound of formula I. ##STR11##

The preparation of ether derivatives of sordarin where R¹² is either--CH═NOH or --CH₂ OCH₃ is shown in Scheme 5. Treatment of the protectedsordarin derivative with a strong base such as sodium hydride and analkyl, alkenyl or aralkyl halide or tosylate under conditions that favoran S_(N) 2 displacement reaction followed by removal of the protectinggroup results in a compound of formula I. ##STR12##

Utility. Compounds of formula I are antifungal agents useful as humanand animal medicaments, as well as crop protectants.

The compounds of formula I are very active fungicides useful incombating fungal infections in animals, including humans. For example,they may be used in the treatment of fungal infections caused byorganisms such as species of Candida (e.g. Candida albicans, Candidaglabrata, (Torulopsis glabrata), Candida tropicalis, and Candidapseudotropicalis), Cryptococcus neoformans, Pneumocystiscarinii,Aspergillus S (e.g. Aspergillus flavus and Aspergillusfumigatus), Coccidioides (e.g. Coccidjoides immitis), Paracoccidioides(e.g. Paracoccidioides brasiliensis), Histoplasma (e.g. Histoplasmacapsulatum) or Blastomyces (e.g. Blastomyces dermatitidis). They mayalso be used to treat other fungal infections caused by species ofTrichophyton, Microsporum or Epidermophyton (e.g. Trichophytonmentographytes, Trichophyton rubrum, Microsporum canis or Epidermophytonfloccosum), or in mucosal infections caused by Candida albicans.

Compounds of formula I may also be used to treat other infections causedby species of filamentous fungi such as Geotrichum (e.g. Geotrichumclavatum), Trichosporon (e.g. Trichosporon beigelii), Blastoschizomyces(e.g. Blastoschizomyces capitatus), Sporothrix (e.g. Sporothrixschenckii), Scedosporium (e.g. Scedosporium apiosperum), Cladosporium(e.g. Cladosporium carrionii) and Pityrosporum ovale.

The compounds of formula I may also be used to treat infections causedby protozoa such as Toxoplasma, Cryptosporidium, Leishmania,Tripanosoma, Giardia and Trichomonas.

The in vitro evaluation of the anti-fungal activity of compounds of theinvention was performed on liquid or solid medium by the anti-fungaltwo-fold serial dilution technique of determining the minimum inhibitoryconcentration (MIC) of anti-fungal agent that inhibited development ofgrowth after 24 to 48 hours of incubation at 35° C. In practice, aseries of agar plates or broth microdilution panels containing two-folddilutions of anti-fungal agent tested were inoculated with a standardculture of a clinically relevant pathogen, for example, Candidaalbicans. The agar plates or broth microdilution panels were thenexamined for the presence or absence of growth of the fungus and theappropriate MIC values were noted. Visualization of endpoints wasassisted by employment of the vital stain Alamar Blue.

The in vivo evaluation of compounds of formula I can be carried out at aseries of dose levels by administration (e.g. subcutaneously, orally,intraperitoneally or intravenously) to mice inoculated intravenouslywith a strain of Candida spp. The kidneys of the test animals may beremoved and quantitated for viable Candida spp. and the reduction ininfection may be determined relative to untreated control animals.

In view of their antifungal activity, compounds of formula I are usefulfor the treatment and/or prevention of a variety of fungal infections inhuman beings and animals. Such infections include superficial,cutaneous, subcutaneous and systemic mycotic infections such asrespiratory tract infections, gastrointestinal tract infections,cardiovascular infections, urinary tract infections, CNS infections,candidiasis and chronic mucocandidiasis (e.g. thrush and vaginalcandidiasis) and skin infections caused by fungi, cutaneous andmucocutaneous candidiasis, dermatophytoses including ringworm and tineainfections, athletes foot, paronychia, pityriasis versicolor,erythrasma, intertrigo, fungal diaper rash, candida vulvitis, candidabalanitis and otitis externa. They may also be used as prophylacticagents to prevent systemic and topical fungal infections. Use asprophylactic agents may, for example, be appropriate as part of aselective gut decontamination regimen in the prevention of infection inimmunocompromised patients (e.g. AIDS patients, patients receivingcancer therapy or transplant patients). Prevention of fungal overgrowthduring antibiotic treatment may also be desirable in some diseasesyndromes or iatrogenic states.

Compounds of formula I also have use as broad spectrum crop antifungalagents and are effective on a broad spectrum of phytopathogenic fungi,in particular those from the class consisting of: Deuteromycetes (e.g.Botrytis spp., Septoria spp., Pyricularia spp., Stagnospora spp.,Helminthosporium spp., Fusarium spp., Cercospora spp., Rhynchosporium,spp. Pseudocercosporella, spp. and Alternaria spp.); Basidiomycetes(e.g. Puccinia spp., Rhizoctonia spp., and Hemileia); Ascomycetes (e.g.Venturia spp., Podospharera spp., Erysiphe spp., Monilinia spp. andUncinula spp.); and Oomycetes (e.g. Phytophthora spp., Pemospora spp.,Bremia spp., Pythium spp., and Plasmopara spp.). The foregoing listexemplifies the phytopathogenic fungi against which the named compoundsdemonstrate activity, and is not limiting in any manner. These compoundshave very advantageous curative and preventive fungicidal properties forprotecting plants, and can be used to inhibit or to destroy themicroorganisms occurring on plants or on parts of plants (the fruit,blossom, leaves, stalks, tubers or roots) of different crops of usefulplants, while at the same time parts of plants that grow later are alsoprotected against such microorganisms. They can also be used asdressings in the treatment of plant propagation material, especiallyseed (fruit, tubers, grain) and plant cuttings (for example rice), toprovide protection against fungal infections and against phytopathogenicfungi occurring in the soil. Compounds of formula I of the invention aredistinguished by the fact that they are especially well tolerated byplants and are environmentally friendly.

Agricultural evaluation of compounds of formula I can be carried outusing the following tests.

1. Action Against Erysiphe graminis on wheat

a) After 1 week cultivation, wheat plants are sprayed to run off with aspray mixture (200 ppm active ingredient/20% acetone/0.25% Triton X155).After 2 hours, the treated plants are infected with ascospores shakenfrom inoculum plants. Fungal attack is evaluated after incubation for 8days at 22° C. at 50% relative humidity to determine the protectiongiven by the compound.

b) After 1 weeks cultivation, wheat plants are infected with ascosporesshaken from inoculum plants. After 24 hours, the wheat plants aresprayed with a spray mixture (200 ppm active ingredient/20%acetone/0.25% Triton X155). Fungal attack is evaluated after incubationfor 8 days at 22° C. at 50% relative humidity to determine the degree ofcurative activity provided by the compound.

c) After 1 weeks cultivation, wheat plants are infected with ascosporesshaken from inoculum plants. After 24 hours, the soil in which the wheatplants are growing is drenched with the drench mixture (200 ppm activeingredient/20% acetone/0.25% Triton X155). Fungal attack is evaluatedafter incubation for 8 days at 22° C. at 50% relative humidity todetermine the degree of curative activity provided by the compound.

2. Action Against Puccinia recondita on wheat

a) After 1 weeks cultivation, wheat plants sprayed to run off with aspray mixture (200 ppm active ingredient/20% acetone/0.25% Triton X155).After 2 hours, the treated plants are infected with a spore. Fungalattack is evaluated after incubation for 1 day at 95-100% relativehumidity at 20° C. followed by 7 days at 25° C. at 50% relative humidityto determine the protection given by the compound.

b) After 1 weeks cultivation, wheat plants are infected with a sporesuspension After 24 hours, the infected plants are sprayed to run offwith a spray mixture (200 ppm active ingredient/20% acetone/0.25% TritonX155. Fungal attack is evaluated after incubation for 1 day at 95-100%relative humidity at 20° C. followed by 7 days at 25° C. at 50% relativehumidity to determine the degree of curative activity provided by thecompound.

c). After 1 weeks cultivation, wheat plants are infected with a sporesuspension After 24 hours, the soil in which the wheat plants aregrowing was drenched with the drench mixture (200 ppm activeingredient/20% acetone/0.25% Triton X155). Fungal attack is evaluatedafter incubation for 1 day at 95-100% relative humidity at 20° C.followed by 7 days at 25° C. at 50% relative humidity to determine thedegree of curative activity provided by the compound.

Based on the spectrum of activity, the compounds of the presentinvention can be used to protect or cure plants of phytopathogenic fungiaffecting various useful crops. The following species of plants aresuitable for the use described in the scope of the invention of thestated compounds: cereal (e.g. wheat, rye, oat, barley, rice, sorghumand related crops); beet (sugar beet and fodder beet); pomes, dropes andsoft fruit (e.g. apples, pears, plums, peaches, almonds, cherries,strawberries, raspberries, and blackberries); leguminous plants (e.g.beans, peas, lentils and soybeans); oil plants (rape, mustard, poppy,olives, sunflowers, coconut, castor oil plants, cocoa beans andgroundnuts); curbitats (e.g. cucumber, squash, and melon); fiber plants(e.g. cotton, flax, hemp, and jute); citrus fruit (e.g. oranges, lemons,madarins and grapefruit); vegetables (e.g. lettuce, cabbage, spinach,carrot, asparagus, paprika, onions, tomatoes, and potatoes); lauraceae:(avocados, cinnamon and camphor); or plants such as maize, tobacco,nuts, coffee, sugar cane, tea, vines, hops, bananas and natural rubberplants, as well as ornamentals (flowers, shrubs, broad-leaved trees andevergreens, such as conifers). However, the aforementioned plant speciesdo not constitute a limiting list of plants with respect to spectrum bythe stated compounds.

The compounds of formula I are particularly useful for controlling thefollowing plant diseases:

Erysiphe graminis in cereals, Erysiphe cichoracearum and Sphaerothecafuliginea in cucurbits, Podosphaera leucotricha in apples, Uncinulanecator in vines, Puccinia species in cereals, Rhizoctonia solani incotton, Ustilago species in cereals and sugar cane, Venturia inaequalis(scab) in apples, Helminthosporium species in cereals, Septoria nodorumin wheat, Botrytis cinerea (gray mold) in strawberries and grapes,Cercospora arachidicola in groundnuts, Pseudocercosporellaherpotrichoides in wheat and barley, Pyricularia oryzae in rice,Phytophthora infestans in potatoes and tomatoes, Fusarium andVerticillium species in various plants, Plasmopara viticola in grapes,Altemaria species in fruit and vegetables. The compounds of formula Imay also be used for protecting materials (e.g. preservation of timberagainst Paecilomyces variotii).

Pharmaceutical Compositions. While it is possible that, for use intherapy, compounds of the invention may be administered as the rawchemical, it is preferable to present the active ingredient in apharmaceutical composition. The invention thus further provides apharmaceutical composition comprising a compound of formula (I) or apharmaceutically acceptable salt thereof, together with one or morepharmaceutically acceptable carriers thereof and, optionally, othertherapeutic and/or prophylactic ingredients. The carrier(s) must be`acceptable` in the sense of being compatible with the other ingredientsof the formulation and not deleterious to the recipient thereof.

The compositions of the invention include those in a form especiallyformulated for oral, buccal, parenteral, implant, rectal, topical,ophthalmic or genito-urinary administration or in a form suitable foradministration by inhalation or insufflation.

Tablets and capsules for oral administration may contain conventionalexcipients such as binding agents, for example, syrup, acacia, gelatin,sorbitol, tragacanth, mucilage of starch or polyvinylpyrrolidone;fillers, for example, lactose, sugar, microcrystalline cellulose,maize-starch, calcium phosphate or sorbitol; lubricants, for example,magnesium stearate, stearic acid, talc, polyethylene glycol or silica;disintegrants, for example, potato starch or sodium starch glycollate orcrosscarmellose sodium; or wetting agents such as sodium laurylsulphate. The tablets which include chewable, dispersible oreffervescent tablets may be coated according to methods well known inthe art. Oral liquid preparations may be in the form of, for example,aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, ormay be presented as a dry product for constitution with water or othersuitable vehicle before use. Such liquid preparations may containconventional additives such as suspending agents, for example, sorbitolsyrup, methyl cellulose, glucose/sugar syrup, gelatin,hydroxyethylcellulose, carboxymethyl cellulose, aluminium stearate gelor hydrogenated edible fats; emulsifying agents, for example, lecithin,sorbitan mono-oleate or acacia; non-aqueous vehicles (which may includeedible oils), for example, almond oil, fractionated coconut oil, oilyesters, propylene glycol or ethyl alcohol; and preservatives, forexample, methyl or propyl p-hydroxybenzoates or sorbic acid.

For buccal administration the composition may take the form of tabletsor lozenges formulated in conventional manner.

The composition according to the invention may be formulated forparenteral administration by injection or continuous infusion.Formulations for injection may be presented in unit dose form inampoules, or in multi-dose containers with an added preservative. Thecompositions may take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and may contain formulatory agents such assuspending, stabilizing and/or dispersing agents. Alternatively theactive ingredient may be in powder form for constitution with a suitablevehicle, e.g. sterile, pyrogen-free water, before use.

For administration by inhalation the compositions according to theinvention are conveniently delivered in the form of an aerosol spraypresentation from pressurized packs with the use of a suitablepropellant, e.g. dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas, or froma nebuliser. In the case of a pressurized aerosol the dosage unit may bedetermined by providing a valve to deliver a metered amount.

Alternatively, for administration by inhalation the compositionsaccording to the invention may take the form of a dry powdercomposition, for example a powder mix of the compound and a suitablepowder base such as lactose or starch or as a modified physical form ofthe drug substance alone. The powder composition may be presented inunit dosage form in, for example, capsules or cartridges of e.g.gelatin, or blister packs from which the powder may be administered withthe aid of an inhaler or insufflator.

The compositions may take the form of a suppository, e.g. containing aconventional suppository base, or a pessary, e.g. containing aconventional pessary base.

The compositions may also be formulated for topical administration inthe form of ointments, creams, gels, lotions, shampoos, powders(including spray powders), pessaries, tampons, sprays, dips, aerosols,drops (e.g. eye, ear or nose drops) or pour-ons. Ointments and creamsmay, for example, be formulated with an aqueous or oily base with theaddition of suitable thickening and/or gelling agents. Ointments foradministration to the eye may be manufactured in a sterile manner usingsterilized components. Pour-ons may, for example, be formulated forveterinary use in oils containing organic solvents, optionally withformulatory agents, e.g. stabilizing and solubilizing agents. Pessariesand tampons for vaginal insertion may be formulated using conventionaltechniques and, where appropriate, may contain an effervescent vehicle.Such compositions may also contain other active ingredients such ascorticosteroids, antibiotics or antiparasitics as appropriate.

Liquid preparations for intranasal delivery may take the form ofsolutions or suspensions and may contain conventional excipients such astonicity adjusting agents, for example, sodium chloride, dextrose ormannitol; preservatives, for example benzalkonium chloride, thiomersal,phenylethyl alcohol; and other formulating agents such as suspending,buffering, stabilizing, dispersing and or flavouring agents.

Transdermal administration may be affected by the design of a suitablesystem which promot-es absorption of the active compound through theskin and would typically consist of a base formulation enclosed withinan adhesive stick-on patch comprising backing films, membranes andrelease liners. Such systems may include absorption enhancers such asalcohols or work by promoting ionotophoresis.

The composition according to the invention may also be formulated as adepot preparation. Such long acting formulations may be administered byimplantation (for example subcutaneously or intramuscularly) or byintramuscular injection. Thus, for example, a compound of the inventionmay be formulated with suitable polymeric or hydrophobic materials (forexample as an emulsion in an acceptable oil) or ion exchange resins, oras sparingly soluble derivatives, for example, as a sparingly solublesalt.

When the compositions comprise dosage units, each unit will preferablycontain 0.001 mg to 1000 mg, advantageously 0.01 mg to 400 mg, of activeingredient where a compound of the invention is to be administeredorally. The daily dosage as employed for adult human treatment willpreferably range from 0.001 mg to 5000 mg of active ingredient, mostpreferably from 0.01 mg to 2000 mg which may be administered in 1 to 4daily doses, for example, depending on the route of administration andon the condition of the patient and the disease to be treated.

The compound may be administered by intravenous infusion using, forexample, up to 50 mg/kg/day of the active ingredient. The duration oftreatment will be dictated by the rate of response rather than byarbitrary number of days.

Compounds of the invention may also be used in combination with othertherapeutic agents, and the invention thus provides, in a furtheraspect, a combination comprising a compound of the invention togetherwith another therapeutically active agent.

Thus, for example the compounds of the invention may be used incombination with one or more other antifungal agents, such as a polyenicderivative e.g. (Amphotericin B, Nystatin, a lipid formulation ofAmphotericin B) an azole derivative e.g. (Fluconazole, Intraconazole,Ketoconazole, Miconazole, Clotrimazole, ZD-08070, UK-109496, SCH 56592),5-Fluorocytosine, a Pneumocandin or Echinocandin derivative such asCilofungin, LY-303366, L-733560, L-743872 or other cell wall activecompound such as Nikkomycin Z and/or one or more immunomodulating agentssuch as an interferon e.g. (IFN-), interleukine e.g. (IL-1, IL-2, IL-3and IL-8) and colony stimulating factors, [(G)-CSF, (M)-CSF and(GM)-CSF] and defensines. Particularly advantageous compounds for usewith compounds of the invention include Intraconazole, Flucytosine,Fluconazole or Amphotericin B.

When the compounds of the invention are administered in combination withanother antifungal agent the compounds of the invention and the otherfungal agent can be administered at the recommended maximum clinicaldosage or at lower doses.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus pharmaceuticalformulations comprising a combination as defined above together with apharmaceutically acceptable carrier thereof comprise a further aspect ofthe invention. The individual components of such combinations may beadministered either sequentially or simultaneously in separate orcombined pharmaceutical formulations.

When a compound of the invention is used in combination with a secondtherapeutic agent against the same condition the dose of each compoundmay differ from that when the compound is used alone. Appropriate doseswill be readily appreciated by those skilled in the art.

Agrochemical Compositions. The compounds of formula I can be used ineither an unmodified form or preferably together with adjuvantsconventionally employed in the art of agrochemical formulation and arefor this purpose forms known mainly as: emulsifiable concentrates,coatable pastes, directly sprayable or dilutable solutions, dilutesolution, suspensions (including high-percentage aqueous, oily or othersuspensions), dispersions, oil dispersions, broadcasting agents,wettable powders, soluble powders, dusts, granules, and encapsulations.The formulations are prepared in known manner, e.g. by homogeneouslymixing and/or grinding the active ingredients with extenders, e.g.solvents, solid carriers and, where appropriate, surface-activecompounds (surfactants). Powders, dusts and broadcasting agents may beprepared by mixing or grinding the active ingredients with a solidcarrier. Granules, e.g., coated, impregnated or homogeneous granules,may be prepared by bonding the active ingredients to solid carriers.

Suitable solvents are: aromatic hydrocarbons, preferably the fractionscontaining 8 to 12 carbon atoms, such as xylene mixtures or substitutednaphthalenes, chlorinated aromatics such as chlorobenzenes, phthalates,such as dibutyl or dioctyl phthalate, aliphatic hydrocarbons, such ascyclohexane or paraffins, alcohols and glycols and their ethers andesters, such as ethanol, ethylene glycol, ethylene glycol monomethyl ormonoethyl ether, ketones such as cyclohexanone, amines such asethanolamine, strongly polar solvents, such as N-methyl-2-pyrrolidone,dimethyl sulfoxide or dimethylformamide, and vegetable oils orepoxidised vegetable oils, such as epoxidised coconut oil or soybeanoil; and water.

Examples of surfactants are: alkali metal, alkaline earth metal andammonium salts of aromatic sulfonic acids, e.g., ligninsulfonic acid,phenolsulfonic acid, naphthalenesulfonic acid anddibutylnaphthalenesulfonic acid, and of fatty acids, alkyl and alkylarylsulfonates, and alkyl, lauryl ether and fatty alcohol sulfates, andsalts of sulfated hexadecanols, heptadecanols, and octadecanols, saltsof fatty alcohol glycol ethers, condensation products of sulfonatednaphthalene and naphthalene derivatives with formaldehyde, condensationproducts of naphthalene or naphthalenesulfonic acids with phenol andformaldehyde, polyoxyethylene octylphenol ethers, ethoxylatedisooctylphenol, ethoxylated octylphenol and ethoxylated nonylphenol,alkylphenol polyglycol ethers, tributylphenyl polyglycol ethers,alkylaryl polyether alcohols, isotridecyl alcohol, fatty alcoholethylene oxide condensates, ethoxylated castor oil, polyoxyethylenealkyl ethers, ethoxylated polyoxypropylene, lauryl alcohol polyglycolether acetal, sorbitol esters, lignin-sulfite waste liquors and methylcellulose.

Examples of solid carriers are mineral earths such as silicic acids,silica gels, silicates, talc, kaolin, attapulgus clay, limestone, lime,chalk, bole, loess, clay, dolomite, diatomaceous earth, aluminas calciumsulfate, magnesium sulfate, magnesium oxide, ground plastics,fertilizers such as ammonium sulfate, ammonium phosphate, ammoniumnitrate, and ureas, and vegetable products such as grain meals, barkmeal, wood meal, and nutshell meal, cellulosic powders, etc.

Compounds of formula I may be mixed and applied together with otheractive ingredients, for example herbicides, insecticides, bactericides,nematocides, molluscicides, growth regulators, micronutrients, andfertilizers. The other ingredients may also be one or more fungicidesbelonging to but not restricted to the following classes of fungicides:carboxamides, benzimidazoles, triazoles, hydroxypyridines,dicarboxamides, phenylamides, thiadiazoles. carbamates, cyano-oximes,cinnamic acid derivatives, morpholines, imidazoles, B-methoxy acrylatesand pyridines/pyrimidines. Furthermore, these additional activeingredients may be used as mixtures of several of the preparations, ifdesired together with other application promoting adjuvants usually usedin the art of formulation. Suitable carriers and adjuvants can be solidor liquid and correspond to the substances typically used in formulationtechnology (e.g. natural or regenerated mineral substances, solvents,disperants, and wetting agents).

The following list of fungicides with which compounds of formula I maybe combined is intended to illustrate possible combinations but not toimpose any restrictions. Examples of fungicides which may be combinedwith compounds of formula I are: sulfur, dithiocarbamates and theirderivatives, such as ferric dimethyldithiocarbamate, zincdimethyldithiocarbamate, zinc ethylenebisdithiocarbamate, manganeseethylenebisdithiocarbamate, manganese zincethylenediaminebisdithiocarbamate, tetramethylthiuram disulfides,ammonia complex of zinc N,N'-ethylenebisdithiocarbamate, ammonia complexof zinc N,N'-propylenebisdithiocarbamate, zincN,N'-propylenebisdithiocarbamateand N,N'-polypropylenebis (thiocarbamyl)disulfide; nitro derivative, such as dinitro(1-methylheptyl)-phenylcrotonate, 2-sec-butyl-4,6-dinitrophenyl3,3-dimethylacrylate,2-sec-butyl-4,6-dinitrophenyl isopropylcarbonateand diisopropyl 5-nitroisophthalate; heterocyclic substances, such as2-heptadecylimidazol-2-yl acetate,2,4-dichloro-6-(o-chloroanilino)-s-triazine, O,O-diethylphthalimidophosphonothioate,5-amino-1-[bis-(dimethylamino)-phosphinyl]-3-phenyl-1,2,4-triazole,2,3-dicyano-1,4-dithioanthraquinone,2-thio-1,3-dithio[4,5-b]quinoxaline, methyl1-(butylcarbamyl)-2-benzimidazolecarbamate,2-methoxycarbonylaminobenzimidazole, 2-(fur-2-yl)-benzimidazole,2-(thiazol-4-yl)benzimidazole,N-(1,1,2,2-tetrachloroethylthio)tetrahydrophthalimide,N-trichloromethylthiotetrahydrophthalimide,N-trichloromethylthiophthalimide,N-dichlorofluoromethylthio-N',N'-dimethyl-N-phenylsulfuric acid diamide,5-ethoxy-3-trichloromethyl-1,2,3-thiadiazole,2-thiocyanatomethylthiobenzothiazole, 1,4-dichloro-2,5-dimethoxybenzene,4-(2-chlorophenylhydrazono)-3-methyl-5-isoxazolone, 2-thiopyridine1-oxide, 8-hydroxyquinoline and its copper salt,2,3-dihydro-5-carboxanilido-6-methyl-1,4-oxathiyne,2,3-dihydro-5-carboxanilido-6-methyl-1,4-oxathiyne 4,4-dioxide,2-methyl-5,6-dihydro-4H-pyran-3-carboxanilide,2-methylfuran-3-carboxanilide, 2,5-dimethylfuran-3-carboxanilide,2,4,5-trimethylfuran-3-carboxanilide,2,5-dimethyl-N-cyclohexylfuran-3-carboxamide,N-cyclohexyl-N-methoxy-2,5-diethylfuran-3-carboxamide,2-methylbenzanilide, 2-iodobenzanilide,N-formyl-N-morpholine-2,2,2-trichloroethylacetal,piperazine-1,4-diylbis-(1-(2,2,2-trichloroethyl)-formamide),1-(3,4-dichloroanilino)-1-formylamino-2,2,2-trichloroethane,2,6-dimethyl-N-tridecylmorpholine and its salts,2,6-dimethyl-N-tridecylmorpholine and its salts,N[3-(p-tert.-butylphenyl)-2-methylpropyl]-cis-2,6-dimethylmorpholine,N-[3-(p-tert.-butylphenyl)-2-methylpropyl]-piperidine,1-2-(2,4-dichlorophenyl)-4-ethyl-1,3-dioxolan-2-yl-ethyl]-1H-1,2,4-triazole,1-[2-(2,4-dichlorophenyl)-4-n-propyl-1,3-dioxolan-2-yl-ethyl]-1H-1,2,4-triazole,N-(n-propyl)-N-(2,4,6-trichlorophenoxyethyl)-N]-imidazolylurea,1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-butan-2-one,1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)-butan-2-ol,alpha -(2-chlorophenyl)-alpha-(4-chlorophenyl)-5-pyrimidinemethanol,5-butyl-(2-dimethylamino-4-hydroxy-6-methylpyrimidine,bis-(p-chlorophenyl)-3-pyridinemethanol,1,2-bis-(3-ethoxycarbonyl-2-thioureido)-benzene,1,2-bis-(3-methoxycarbonyl-2-thioureido)-benzene, and variousfungicides, such as dodecylguanidine acetate,3-[3-(3,5-dimethyl-2-oxycyclohexyl)-2-hydroxyethyl]-glutaramide,hexachlorobenzene, DL-methyl-N-(2,6-dimethylphehyl)-N-fur-2-yl alanate,methyl DL-N-(2,6-dimethylphenyl)-N-(2]-methoxyacetyl)-alanate,N-(2,6-dimethylphenyl)-N-chloroacetyl-DL-2-aminobutyrolactone, methylDL-N-(2,6-dimethylphenyl)-N-(phenylacetyl)-alanate,5-methyl-5-vinyl-3-(3,5-dichlorophenyl)-2,4-dioxo-1,3-oxazolidine,3-[3,5-dichlorophenyl]-5-methyl-5-methoxymethyl-1,3-oxazolidine-2,4-dione,3-(3,5-dichlorophenyl)-1-isopropylcarbamylhydantoin,N-(3,5-dichlorophenyl)-1,2-dimethylcyclopropane-1,2-dicarboximide,2-cyano-[N-(ethylaminocarbonyl)-2-methoximino]-acetamide,1-[2-(2,4-dichlorophenyl)-pentyl]-1H-1,2,4-triazole,2,4-difluoro-a-(1H-1,2,4-triazol-1-ylmethyl)-benzhydryl alcohol,N-(3-chloro-2,6-dinitro-4-trifluoromethylphenyl)-5-trifluoromethyl-3-chloro-2-aminopyridine,and 1-((bis-(4-fluorophenyl)-methylsilyl)-methyl)-1H-1,2,4-triazole.

As with the nature of compositions, the method of application such asspraying, atomizing, dusting, scattering, coating, dressing, and pouringare chosen in accordance with the intended objectives of the applicationand the prevailing circumstances. One method of applying the activeingredient or agrochemical composition containing at least one of thestated compounds is application to the plants (i.e. foliar application).However, the active ingredient can also penetrate the plant through theroots via the soil (i.e. soil application). This may be in the form ofeither a liquid application to the soil (drench) or a granularapplication.

The active ingredient can also be applied to plant propagation materialsuch as seeds (fruits, tubers or grains) or plant cuttings, in eitherliquid form (coating) or in solid form (dressing). Seeds, for example,can be dressed before sowing. The compounds of the inventioncan also beapplied to grains either by impregnating the grains with a liquidformulation of by coating them with a solid formulation. The compositioncan also be applied to the locus of planting when planting thepropagation material, for example to the seed furrow during sowing.

Advantageous rates of application are normally from 10 g to 50 kg ofactive ingredient (a.i.) per hectare, preferably 100 g to 2 kg a.i./ha,most preferably 100 g to 600 g a.i./ha. The active ingredients of thestated compounds are typically used in the form of compositions and canbe applied to the plant, or to parts of the plant either simultaneouslyor in succession with further active ingredients. These further activeingredients can be fertilizers, additional micronutrients, or otherplant growth affecting compounds. They can, however, also be selectiveherbicides, insecticides, bactericides, nematocides, insecticides, andmolluscicides, as well as other fungicides.

PREPARATION OF STARTING MATERIAL

Fermentation Production of Sordarin

The following media are used in the fermentation of Roselliniasubiculata (ATCC 74386) and ATCC 74387 in the production of sordarin.

SEED MEDIUM 1

    ______________________________________                                               Component                                                                             g/L                                                            ______________________________________                                               Yeast extract                                                                         4.0                                                                   Malt extract                                                                          8.0                                                                   Glucose 4.0                                                                   Junlon  1.5                                                            ______________________________________                                    

The medium was prepared with distilled water, the pH adjusted to 7.0prior to sterilization, and was dispensed at 50 ml/250 ml unbaffledErlenmeyer flask. Cotton closures were used. Sterilization was at 121°C. for 20 minutes.

SEED MEDIUM 2

    ______________________________________                                                        Trace elements solution                                       Component    (g/l)    Component      (g/l)                                    ______________________________________                                        Corn steep liquor (dried)                                                                   2.5     FeSO.sub.4.7H.sub.2 O                                                                        1.0                                      Tomato paste 40.0     MnSO.sub.4.4H.sub.2 O                                                                        1.0                                      Oat flour    10.0     CuCl.sub.2.2H.sub.2 O                                                                        0.025                                    Glucose      10.0     CaCl.sub.2.H.sub.2 O                                                                         0.1                                      Trace elements                                                                             10.0 ml/L                                                                              H.sub.3 BO.sub.3                                                                             0.056                                    solution              (NH.sub.4).sub.6 MoO.sub.24.4H.sub.2 O                                                       0.019                                                          ZnSO.sub.4.7H.sub.2 O                                                                        0.2                                                            Trace elements prepared                                                       in 0.6N HCl                                             ______________________________________                                    

The medium was prepared with distilled water, the pH adjusted to 6.8prior to sterilization, and was dispensed at 50 ml/250 ml unbaffledErlenmeyer flask. Cotton closures were used. Sterilization was at 121°C. for 20 minutes.

Solid Production Medium 1

1. Solid Portion

Add 675 cc vermiculite to a 2-liter roller bottle. Plug with latexclosure; autoclave for 60 min., plus 30 min. dry.

2. Liquid Portion

To a 500 ml bottle, add 220 ml of the following:

    ______________________________________                                        Component               g/L                                                   ______________________________________                                        Glucose                150.0                                                  Glycerol                20.0                                                  Yeast extract           4.0                                                   NaNO.sub.3              1.0                                                   Monosodium Glutamate    3.0                                                   Na.sub.2 HPO.sub.4      0.5                                                   MgSO.sub.4.7H.sub.2 O   1.0                                                   K-elements              1.0 ml/L                                              CaCO.sub.3              8.0                                                   ______________________________________                                        K-elements                                                                    Component               (g/l)                                                 ______________________________________                                        FeCl.sub.3.6H.sub.2 O   5.8                                                   MnSO.sub.4.H.sub.2 O    0.1                                                   CoCl.sub.2.6H.sub.2 O   0.02                                                  CuSO.sub.4.5H.sub.2 O   0.015                                                 Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                         0.012                                                 ZnCl.sub.2              0.02                                                  SnCl.sub.2.2H.sub.2 O   0.005                                                 H.sub.3 BO.sub.3        0.01                                                  KCl                     0.02                                                  HCl (concentrated)      2.0 ml/L                                              ______________________________________                                    

The medium was prepared with distilled water, pH to 7.0 prior tosterilization. Glucose was autoclaved separately. It was dispensed in500 ml bottles and autoclaved at 121° C. for 15 minutes.

Liquid Production Medium 1

    ______________________________________                                               Component g/L                                                          ______________________________________                                               Glycerol  75.0                                                                Glucose   75.0                                                                Tomato paste                                                                            5.0                                                                 NZ amine Type A                                                                         4.0                                                                 Ardamine PH                                                                             5.0                                                                 K.sub.2 HPO.sub.4                                                                       0.5                                                                 MgSO.sub.4.7H.sub.2 O                                                                   0.25                                                                KCl       0.25                                                                ZnSO.sub.4.7H.sub.2 O                                                                   0.5                                                                 CaCO.sub.3                                                                              10.0                                                         ______________________________________                                    

The medium was prepared with distilled water, pH to 7.0 prior tosterilization. The medium was dispensed at 50 ml per 250 ml unbaffledErlenmeyer flask. The flasks were closed with cotton and autoclaved at121° C. for 20 minutes.

Solid Production Medium 2

    ______________________________________                                        Component             g/L                                                     ______________________________________                                        Sucrose               60.0                                                    Glucose               80.0                                                    Glycerol              60.0                                                    Citric Acid           15.0                                                    NZ amine Type A        5.0                                                    NaNO.sub.3             1.0                                                    KH.sub.2 PO.sub.4      0.5                                                    MgSO.sub.4.7H.sub.2 O  0.5                                                    CaCO.sub.3             0.5                                                    K-elements             1 ml/L                                                 ______________________________________                                        K-elements                                                                    Component             (g/l)                                                   ______________________________________                                        FeCl.sub.3.6H.sub.2 O  5.8                                                    MnSO.sub.4.H.sub.2 O   0.1                                                    CoCl.sub.2.6H.sub.2 O  0.02                                                   CuSO.sub.4.5H.sub.2 O  0.015                                                  Na.sub.2 MoO.sub.4.2H.sub.2 O                                                                        0.012                                                  ZnCl.sub.2             0.02                                                   SnCl.sub.2.2H.sub.2 O  0.005                                                  H.sub.3 BO.sub.3       0.01                                                   KCl                    0.02                                                   HCl (concentrated)     2.0 ml/L                                               ______________________________________                                    

The medium was prepared with distilled water, pH to 7.0 prior tosterilization. It was dispensed at 220 ml per 500 ml bottle andautoclaved at 121° C. for 15 minutes.

Liquid Production Medium 2

The composition is the same as the liquid portion of Solid ProductionMedium 1. The medium was prepared with distilled water, pH to 7.0 priorto sterilization. Glucose was autoclaved separately. The medium wasdispensed at 50 ml per 250 ml unbaffled Erlenmeyer flask. The flaskswere closed with cotton and autoclaved at 121° C. for 15 minutes.

Production of Sordarin bv Fermentation of Rosellina subiculata (MF6239.ATCC 74386)

1. CULTURE: A portion of the agar slant containing the culture wasaseptically transferred to seed medium 1 (50 ml/250 ml unbaffled flask).This was incubated on a 2-inch throw gyratory shaker, 220 rpm for 5 daysat 25° C., 85% relative humidity (rh), to obtain biomass. Portions ofthe biomass were transferred into sterile vials containing glycerol andfrozen (as frozen vegetative mycelia (FVM)). These were maintained in afinal concentration of 10-15% glycerol at -75° C. Secondary FVMs wereprepared from a primary FVM by transferring 1.0 ml of the thawed primaryFVM into seed medium 2, incubating 7 days at 25° C., 220 rpm andfreezing as above.

2. SEED: A frozen vial (FVM) of MF6239 was thawed to room temperatureand used to inoculate seed cultures with 1.0 ml per 50 ml seed medium 2.These were grown on a gyratory shaker (220 rpm) for 7 days at 25° C.,85% rh.

3. PRODUCTION: On solid production medium. An aliquot (10-12 ml) of theseed was placed into 220 ml of the liquid portion of solid productionmedium 1. This flask was swirled vigorously to disperse the biomass. Thecontents were dispensed by pouring into a 2 L roller culture vesselwhich contained 675 cubic centimeters of large-particle vermiculite. Thecontents of the roller bottle were shaken/mixed to insure homogeneousinoculation and coverage. The roller bottles were incubatedhorizontally, revolving at approximately 4 rpm on a Wheaton rollerapparatus, at 22° C., 70% rh for 17 days, to obtain a secondarymetabolite in the fermentation medium.

In liquid production medium. Seed cultures were inoculated as describedabove. An aliquot of the seed (1.5 ml) was used to inoculate eachproduction flask, containing 50 ml/250 ml flask of liquid productionmedium 1. Flasks were incubated on a gyratory shaker (220 rpm) for 7-21days at 25° C., 50-85% rh.

Production of Sordarin bv Fermentation of MF6232 (ATCC 74387)

1. CULTURE: A portion of the agar slant containing MF6232 wasaseptically transferred to seed medium 1 (50 ml/250 ml unbaffled flask).This was incubated on a 2-inch throw gyratory shaker, 220 rpm for 3 daysat 25° C., 85% relative humidity (rh), to obtain biomass. Portions ofthe biomass were transferred into sterile vials containing glycerol andfrozen (as FVM). These were maintained in a final concentration of10-15% glycerol at -75° C. Secondary FVMs were prepared from a primaryFVM by transferring 1.0 ml of the thawed primary FVM into seed medium 2(composition below), incubating 7 days, 25° C., 220 rpm, and freezing asabove.

2. SEED: A frozen vial (FVM) of MF6232 was thawed to room temperatureand used to inoculate seed cultures with 1.0 ml per 50 ml seed medium 2.These were grown on a gyratory shaker (220 rpm) for 7 days at 25° C.,85% rh.

3. PRODUCTION: On solid production medium. An aliquot (10-12 ml) of theseed was placed into 220 ml of solid production medium 2. This wasswirled vigorously to disperse the biomass. The contents were dispensedby pouring into a 2 L roller culture vessel which contained 675 cubiccentimeters of large-particle vermiculite. The contents of the rollerbottle were shaken/mixed to insure homogeneous inoculation and coverage.The roller bottles were incubated horizontally, revolving atapproximately 4 rpm on a Wheaton roller apparatus, at 22° C., 70% rh for21 days, to obtain a secondary metabolite in the fermentation medium.

In liquid production medium. Seed cultures were inoculated as describedabove. An aliquot of the seed (1.5 ml) was used to inoculate eachproduction flask, containing 50 ml/250 ml flask of liquid productionmedium 2. Flasks were incubated on a gyratory shaker (220 rpm) for 7-21days at 25° C., 50-85% rh.

Large Scale Production of Sordarin by MF6232 (ATCC 74387)

The liquid portion of solid production medium 1 was used for both theseed and production fermenters. Cerelose, added post-sterilely, in theseed fermenter medium was 30 g/L while that of the production fermentermedium was 150 g/L. Seed fermenters were inoculated with 2 L of culturegrown in shaker flasks. These fermenters were permitted to grow at 25°C. for 30 hours until the oxygen uptake rate was about 3 mmol/L-hr. At30 hours, 25 L of fermenter seed culture was transferred to theproduction fermenter.

Growth in the production fermenter reached 8-10 mmol/L-hr after 50 hoursand declined to between 5-7 by the end of the cultivation. Dissolvedoxygen was controlled by increasing agitation. Broth pH was notcontrolled and generally decreased to 5.3 at 200 hours. The temperaturewas 25° C.

After 280 hours of growth the fermentation was terminated and thepreparations for harvest begun. The pH was adjusted to about 12 withsodium hydroxide and the batch aged for 20 hours at fermentationtemperature. The pH was then adjusted to 6.0 with sulfuric acid prior totransfer into drums for further processing.

Isolation of Sordarin

Isolation I

A methyl ethyl ketone extract of the fermentation of culture MF6232(ATCC 74387) corresponding to 64 mL of whole broth was concentrated todryness in vacuo (365 mg). This material was dissolved in 2 partsmethanol in 98 parts methylene chloride to a final volume of 4.6 ml. A4.3 ml portion (341 mg) was applied to a 60 ml silica gel 60(0.040-0.0630 mm, 230-400 mesh, E. Merck ) flash chromatography columnequilibrated with 2 percent methanol in methylene chloride. The columnwas eluted by a step gradient of 240 ml each of 2, 5, 10, and 30 percentmethanol in methylene chloride followed by 120 ml of methanol. Sixteen15 ml fractions were collected from each solvent system. The productrich fractions 39-56 were determined by biological assay.

The crude fraction pool was concentrated to dryness in vacuo (103.1 mg).A 34.4 mg portion of this sample was further purified by HPLC separation(Zorbax Rx-C₈, 5 μm, 9.4 mm×250 mm, eluted with mobile phase consistingof 20% acetonitrile/80% aqueous 0.01 M K₂ HPO₄ adjusted to pH 6.9 withconcentrated H₃ PO₄, flow rate 4 ml/min. at 40° C., diode arraydetection). Four milliliter fractions were collected. The product richfractions 16-20 were pooled and concentrated in vacuo to approximatelytwenty-five percent of the original volume. The concentrate was doublyextracted with an equal volume of ethyl acetate and the ethyl acetatelayers were washed with an equal volume of brine, dried over anhydrousNa₂ SO₄ and concentrated in vacuo to yield 3.7 mg of sordarin.

Isolation II

A methyl ethyl ketone extract of the batch -004Y fermentation of cultureMF6232 (ATCC₇₄₃₈₇) corresponding to 980 mL of whole broth wasconcentrated to dryness in vacuo (4.9 g). This material was dissolved in1 part methanol in 9 parts methylene chloride to a final volume of 21.5ml. A 21 ml portion (4.8 g) was applied to a 500 milliliter silica gel60 (0.040-0.0630 mm, 230-400 mesh, E. Merck) chromatography columnequilibrated with 2 percent methanol in methylene chloride. The columnwas eluted at a flowrate of 25 ml/min. by a step gradient beginning with1 liter each of 2 and 5 percent methanol in methylene chloride followedby 2 liters of 15 percent methanol. The column elution was completedwith 1 liter each of 30 and 100 percent methanol. Twenty-five milliliterfractions were collected. Product rich fractions 75-85 and 111-121 weredetermined by biological assay and contained Compound I by RP HPLCanalysis under acidic conditions.

The crude fraction pools, 75-85 and 111-121 were concentrated,separately, to dryness in vacuo (69.3 mg and 95.3 mg, respectively). Two34 mg portions of pool 75-85 were further purified by two identical HPLCseparations (Zorbax Rx-C₈, 7 μm, 21.2 mm×250 mm, eluted with mobilephase consisting of 40% acetonitrile/60% H₂ O with 0.1% H₃ PO₄ overall,flow rate 20 ml/min. at 25° C., 220 nm). Ten milliliter fractions werecollected. The product rich fractions 27-31 from both runs were pooledtogether and concentrated in vacuo to approximately forty percent of theoriginal volume. The concentrate was extracted with an equal volume ofethyl acetate and washed with an equal volume of brine, dried overanhydrous Na₂ SO₄ and concentrated in vacuo to yield 27 mg of sordarin.Two 46 mg portions of pool 111-121 were also further purified under theidentical HPLC conditions listed above. Fractions 25-28 from both runswere combined and prepared as described above to yield an additional 17mg of sordarin.

Preparation of Sordaricin benzyl ester

Sordarin (2 mg) was dissolved in 1 mL of acetone. Concentrated HCl (0.2mL) was added. The mixture was stirred at room temperature for 1 day.After dilution with water and aqueous work-up (CH₂ Cl₂), the organicfraction was dried over Na₂ SO₄, filtered and concentrated in vacuo. Themixture was dissolved in 2 mL of DMF to which was added 0.1 mL of benzylbromide, followed by excess solid NaHCO₃. The mixture was stirred atroom temperature overnight, and was then concentrated in vacuo.Chloroform was added to the mixture which was filtered to remove theNaHCO₃. The filtrate was concentrated in vacuo and purified bypreparative thin layer chromatogrpahy (PTLC) to yield 1.0 mg ofsordaricin benzyl ester. ¹ H NMR (CDCl₃): δ0.51 (3H, d, J=6.9), 0.82(3H, d, J=6.6), 0.91 (1H, m), 1.0 (3H, d, J=6.6), 1.18 (1H, d, J=12.6),1.50-2.00 (9H, m), 2.24 (1H, m), 2.51 (1H, m), 3.48 (1H, d, J=11.0),3.87 (1H, d, J=11.0), 5.11 (1H, d, J=11.7), 5.31 (1H, d, J=11.7), 6.04(1H, d, J=2.1), 7.31-7.40 (5H, m), 9.62 (1H, s).

Preparation of Sordaricin p-methoxybenzvl ester

The same procedure for the preparation of sordaricin benzyl ester wasfollowed, with the use of 4-methoxybenzyl chloride instead of benzylbromide. ¹ H NMR (CDCl₃): δ0.51 (3H, d, J=6.9), 0.82 (3H, d, J=6.9),1.00 (3H, d, J=6.9), 0.90-2.00 (11H, m), 2.23 (1H, m), 2.49 (1H, t,J=3.8), 3.79 (3H, s), 4.61 (2H, s), 5.05 (1H, d, J=11.7), 5.26 (1H, d,J=11.7), 6.03 (IH, d, J=3.2), 6.88 (2H, d, J=8.7), 7.28 (2H, d, J=8.7),9.60 (1H, s).

Preparation of Sordaricin allyl ester

A similar procedure for the preparation of sordaricin benzyl ester isfollowed, with the use of allyl bromide instead of benzyl bromide. Inthis manner, the title compound may be obtained.

Preparation of Sordaricin

To a MeOH solution of sordaricin benzyl ester (0.6 mg) was addedPearlman's catalyst. The mixture was stirred under hydrogen (balloonpressure) for 15 minutes. After filtration through cotton wool andconcentration in vacuo, 0.4 mg of sodaricin was obtained. ¹ H NMR(CDCl₃): δ0.82 (3H, d, J=6.8), 0.98 (3H, d, J=6.6), 1.01 (3H, d, J=6.9),1,23 (1H, m), 1.25 (1H, d, J=12.6), 1.58-2.10 (9H, m), 2.34 (1H, m),2.41 (1H, t, J=3.6), 3.45 (1H, d, J=11.0), 4.14 (1H, d, J=11.0), 6.05(1H, d, J=3.0), 9.75 (1H, s).

The following examples are provided to more fully illustrate theinvention, and are not to be construed as limiting the scope of theinvention in any manner.

EXAMPLE 1 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(acetyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Sordaricin benzyl ester (1.0 mg) was dissolved in 1 mL of pyridine and 1mL of acetic anhydride. A catalytic amount of4-(N,N-dimethylaminopyridine) (DMAP) was added, and the mixture wasstirred at room temperature for 30 minutes. After concentration in vacuoand purification by PTLC, 1.0 mg of the benzyl ester of the titlecompound was obtained. This compound was dissolved in 1 mL of MeOH towhich Pearlman's catalyst was added. The mixture was stirred underhydrogen (ballooon pressure) for 15 minutes. After filtration throughcotton wool and concentration in vacuo, the title compound was obtained.¹ H NMR (CDCl₃): δ0.77 (3H, d, J=6.6), 0.97 (3H, d, J=6.6), 1.01 (3H, d,J=6.8), 1.22 (1H, m), 1.32 (1H, d, J=12.5), 1.72-2.10 (9H, m), 2.02 (3H,s), 2.33 (1H, m), 2.70 (1H, t, J=3.9), 4.22 (1H, d, J=10.7), 4.28 (1H,d, J=10.7), 6.07 (1H, d, J=2.3), 9.68 (1H s).

EXAMPLE 2 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(undecanoyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a tetrahydrofuran (THF) solution of undecanoic acid (30 mg) was addedNEt₃ (34 μL), followed by 2,4,6-trichlorobenzoyl chloride (25 μL). Themixture was stirred at room temperature for 15 minutes. Sordaricinbenzyl ester (1.0 mg) in 1 mL of THF was then added to this mixture,followed by the addition of DMAP (20 mg). This mixture was stirred atroom temperature for 1 hour. After purification by PTLC, the benzylester of the title compound was obtained. This undecanoate was dissolvedin 1 mL of MeOH to which Pearlman's catalyst was added. The mixture wasstirred under hydrogen (balloon pressure) for 15 minutes. Afterfiltration through cotton wool and concentration in vacuo, the titlecompound was obtained. ¹ H NMR (CDCl₃): δ0.77 (3H, d, J=6.6), 0.86 (3H,t, J=6.8), 0.97 (3h, d, J=6.6), 1.10 (3H, d, J=6.8), 0.96-1.02 (2H, m),1.2-1.3 (16H, m), 1.56-2.10 (9H, m), 2.26 (2H, t, J=7.6), 2.34 (1H, m),2.68 (1H, t, J=3.9), 4.20 (1H, d, J=10.8), 4.31 (1H, d, J=10.8), 6.06(1H, d, J=2.3), 9.68 (1H, s).

EXAMPLE 3 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(propanoyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a CH₂ Cl₂ solution of sordaricin benzyl ester (0.5 mg) was added NEt₃(0.2 mL), followed by propionyl chloride (0.1 mL). A catalytic amount ofDMAP was added. The mixture was stirred at room temperature overnight.After purification by PTLC, the benzyl ester of the title compound wasobtained. This compound was dissolved in 1 mL of MeOH and Pearlman'scatalyst was added. Ths mixture was stirred under hydrogen (balloonpressure) for 15 minutes. After filtration through cotton wool andconcentration in vacuo, the title compound was obtained. ¹ H NMR(CDCl₃): δ0.78 (3H, d, J=6.6), 0.97 (3H, d, J=6.8), 1.01 (3H, d, J=6.9),1.12 (3H, t, J=7.6), 1.21 (1H, m), 1.32 (1H, d, J=12.5), 1.70-2.10 (9H,m), 2.29 (2H, t, J=7.6), 2.32 (1H, m), 2.69 (1H, t, J=3.7), 4.21 (1H, d,J=11.0), 4.32 (1H, d, J=11.0), 6.07 (1H, d, J=3.4), 9.70 (1H, s).

EXAMPLE 4 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(methoxycarbonyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 3 was followed, with the use of methylchloroformate instead of propionyl chloride. ¹ H NMR (CDCl₃): δ0.98 (3H,d, J=6.9), 1.01 (3H, d, J=6.9), 1.18 (3H, d, J=6.9), 1.22 (1H, m), 1.32(1H, d, J=12.5), 1.60-2.10 (9H, m), 2.33 (1H, m), 2.75 (1H, t, J=3.7),3.75 (3H, s), 4.32 (1H, d, J=10.3), 4.39 (1H, d, J=10.3), 6.10 (1H, d,J=2.5), 9.65 (1H, s).

EXAMPLE 5 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(propylaminocarbonyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a CHCl3 solution of sordaricin benzyl ester (0.5 mg) was addedn-propyl isocyanate (0.1 mL) and a catalytic amount of DMAP. The mixturewas refluxed for 4 hours. After concentration in vacuo and purificationby PTLC, the benzyl ester of the title compound was obtained. Thisderivative was dissolved in 1 mL of MeOH and Pearlman's catalyst wasadded. Ths mixture was stirred under hydrogen (balloon pressure) for 15minutes. After concentration in vacuo and purification by PTLC, thetitle compound was obtained. 1H NMR (CDCl₃): δ0.78 (3H, d, J=6.4), 0.90(3H, t, J=7.4), 0.96 (3H, d, J=7.1), 1.00 (3H, d, J=6.7), 1.22 (1H, m),1.31 (1H, d, J=12.5), 1.50 (2H, m), 1.50-2.10 (9H, m), 2.32 (1H, m),2.66 (1H, m), 3.10 (2H, m), 4.22 (1H, d, J=10.5), 4.30 (1H, d, J=10.5),4.60 (1H, br), 6.07 (1H, s), 9.70 (1H, s).

EXAMPLE 6 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(methoxymethoxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a CH₂ Cl₂ solution of sordaricin benzyl ester (0.5 mg) was addeddiisopropylethylamine (0.2 mL), followed by methoxymethyl chloride(MOMCl) (0.1 mL) at 0° C. The mixture was stirred at room temperatureovernight. After concentration in vacuo and purification by PTLC, thebenzyl ester of the title compound was obtained. This derivative wasdissolved in methanol and Pearlman's catalyst (palladium hydroxide oncarbon) was added. Ths mixture was stirred under hydrogen (balloonpressure) until removal of the benzyl group was complete as determinedby TLC. After concentration in vacuo and purification by PTLC, the titlecompound was obtained. ¹ H NMR (CDCl₃): δ0.82 (3H, d, J=6.9), 0.96 (3H,d, J=6.7), 1.02 (3H, d, J=6.9), 1.23 (1H, m), 1.28 (1H, d, J=12.5),1.50-2.10 (9H, m), 2.32 (1H, m), 2.48 (1H, m), 3.35 (3H, s), 3.44 (1H,d, J=9.8), 4.00 (1H, d, J=9.8), 4.61 (2H, AB q, J=6.7), 6.05 (1H, d,J=2.6), 9.83 (1H, s).

EXAMPLE 7 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[((methoxyethoxy)methoxy)-methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 6 was followed, with the use ofmethoxyethoxymethyl chloride instead of MOMCl. ¹ H NMR (CDCl₃): δ0.81(3H, d, J=6.4), 0.96 (3H, d, J=7.4), 1.02 (3H, d, J=6.9), 1.10 (1H, m),1.15 (1H, d, J=6.4), 1.50-2.10 (9H, m), 2.33 (1H, m), 2.45 (1H, t,J=3.7), 3.2-3.6 (6H, m), 3.53 (3H, s), 3.93 (1H, d, J=9.3), 3.99 (1H, d,J=9.3), 6.04 (1H, d, J=2.1), 9.90 (1H, s).

EXAMPLE 8 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(octyloxymethoxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 6 was followed, with the use of chloromethyloctyl ether instead of MOMCl. ¹ H NMR (CDCl₃): δ0.82 (3H, d, J=6.9),0.86 (3H, t, J=7.1), 0.96 (3H, d, J=6.7), 1.02 (3H, d J=6.9), 1.20-1.30(14H, m), 1.5-2.1 (9H, m), 2.32 (1H, m), 2.47 (1H, t, J=3.7), 3.44 (1H,d, J=9.4), 3.49 (2H, t, J=6.6), 3.99 (1H, d, J=9.4), 4.67 (2H, s), 6.05(1H, d, J=2.3), 9.84 (1H, s).

EXAMPLE 9 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(methoxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a dimethylformamide (DMF) solution of sordaricin benzyl ester wasadded excess methyl iodide (0.1 mL), followed by NaH (20 mg of a 60% oildispersion). The mixture was stirred at room temperature overnight.After concentration in vacuo and purification by PTLC, the benzyl esterof the title compound was obtained. This derivative was dissolved inmethanol and palladium hydroxide on carbon (Pearlman's catalyst) wasadded. Ths mixture was stirred under hydrogen (balloon pressure) untilremoval of the benzyl group was complete as determined by TLC. Afterconcentration in vacuo and purification by PTLC, the title compound wasobtained. ¹ H NMR (CDCl₃): δ0.82 (3H, d, J=6.9), 0.96 (3H, d, J=6.6),1.03 (3H, d, J=6.9), 1.20-2.20 (11H, m), 2.33 (1H, m), 2.36 (1H, t,J=3.7), 3.26 (1H, d, J=9.1), 3.38 (3H, s), 3.93 (1H, d, J=9.1), 6.03(1H, d, J=2.8), 9.88 (1H, s).

EXAMPLE 10 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(ethoxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of ethyl iodideinstead of methyl iodide. ¹ H NMR (CDCl₃): δ0.83 (3H, d, J=6.8), 0.95(3H, d, J=6.6), 1.02 (3H, d, J=6.9), 1.22 (3H, t, J=7.0), 1.24 (2H, mn),1.78-2.16 (9H, m), 2.30-2.38 (2H, m), 3.25 (1H, d, J=8.4), 3.54 (2H, m),3.98 (1H, d, J=8.4), 6.02 (1H, m), 9.89 (1H, s).

EXAMPLE 11[1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(propyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of allyl bromideinstead of methyl iodide. ¹ H NMR (CDCl₃): δ0.83 (3H, d, J=6.9), 0.91(3H, t, J=7.5), 0.95 (3H, d, J=6.9), 1.02 (3H, d, J=6.8), 1.24 (1H, m),1.26 (1H, d, J=12.5), 1.50-2.15 (11H, m), 2.33 (1H, m), 2.34 (1H, m),3.23 (1H, d, J=9.2), 3.44 (2H, m), 3.99 (1H, d, J=9.2), 6.02 (1H, d,J=2.1), 9.90 (11H, s).

EXAMPLE 12 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(2-methylpropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of2-bromomethyl-1-propene instead of methyl iodide. ¹ H NMR (CDCl₃): δ0.83(3H, d, J=7.1), 0.89 (3H, d, J=6.7), 0.90 (3H, d, J=6.9), 0.95 (3H, d,J=6.8), 1.23 (1H, m), 1.26 (1H, d, J=12.8), 1.50-2.16 (10H, m), 2.32(1H, m), 2.34 (1H, t, J=3.7), 3.18 (1H, dd, J=6.9, 9.4), 3.21 (1H, d,J=9.4), 3.31 (1H, dd, J=6.4, 9.4), 4.00 (1H, d, J=9.4), 6.02 (1H, dd,J=1.2, 3.2), 9.90 (1H, s).

EXAMPLE 13 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(butyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of 1-iodobutane orcrotyl chloride instead of methyl iodide. ¹ H NMR (CDCl₃): δ0.82 (3H, d,J=7.1), 0.90 (3H, t, J=7.5), 0.95 (3H, d, J=6.7), 1.02 (3H, d, J=6.9),1.20-1.28 (2H, m), 1.34 (2H, m), 1.50-2.16 (11H, m), 2.32 (1H, m), 2.35(1H, t, J=3.7), 3.23 (1H, d, J=9.1), 3.46 (2H, m), 3.98 (1H, d, J=9.1),6.02 (1H, d, J=2.3), 9.89 (1H, s).

EXAMPLE 14 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(pentyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use oftrans-1-bromo-2-pentene or 1-iodopentane instead of methyl iodide. ¹ HNMR (CDCl₃): δ0.83 (3H, d, J=6.9), 0.87 (3H, t, J=7.1), 0.96 (3H, d,J=6.8), 1.02 (3H, d, J=6.9), 1.20-1.34 (6H, m), 1.50-2.16 (11H, m), 2.32(1H, m), 2.34 (1H, t, J=3.7), 3.22 (1H, d, J=9.2), 3.46 (2H, m), 3.98(1H, d, J=9.2), 6.02 (1H, d, J=2.1), 9.90 (1H, s).

EXAMPLE 15 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(hexyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of 1-iodohexaneinstead of methyl iodide. ¹ H NMR (CDCl₃): δ0.83 (3H, d, J=7.1), 0.86(3H, t, J=7.3), 0.95 (3H, d, J=6.6), 1.02 (3H, d, J=6.6), 1.20-1.32 (8H,m), 1.50-2.16 (11H, m), 2.32 (1H, m), 2.34 (1H, t, J=3.7), 3.22 (1H, d,J=9.1), 3.46 (2H, m), 3.98 (2H, d, J=9.1), 6.02 (1H, d, J=2.1), 9.90(1H, s).

EXAMPLE 16 [ 1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(S-2-hydroxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of thebenzenesulfonate of (R)-glycidol instead of methyl iodide. ¹ H NMR(CDCl₃): δ0.82 (3H, d, J=6.8), 0.96 (3H, d, J=6.6), 1.02 (3H, d, J=6.9),1.15 (3H, d, J=6.4), 1.24 (1H, m), 1.27 (1H, d, J=12.6), 1.60-2.10 (9H,m), 2.33 (1H, m), 2.46 (1H, t, J=3.9), 3.34 (1H, dd, J=7.4, 10.0), 3.40(1H, dd, J=3.4, 10.0), 3.43 (1H, d, J=9.4), 3.92 (1H, d, J=9.4), 3.99(1H, m), 6.04 (1H, d, J=2.3), 9.83 (1H, s).

EXAMPLE 17 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(R-2-hydroxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of thebenzenesulfonate of (S)-glycidol instead of methyl iodide. ¹ H NMR(CDCl₃): δ0.82 (3H, d, J=6.8), 0.96 (3H, d, J=6.6), 1.02 (3H, d, J=6.9),1.15 (3H, d, J=6.9), 1.23 (1H, m), 1.26 (1H, t, J=12.6), 1.50-2.10 (9H,m), 2.33 (1H, m), 2.45 (1H, t, J=3.9), 3.30 (1H, dd, J=7.5, 10.0), 3.40(1H, dd, J=3.4, 10.0), 3.52 (1H, d, J=9.4), 3.97 (1H, d, J=9.4), 3.99(1H, m), 6.04 (1H, d, J=2.6), 9.83 (1H, s).

EXAMPLE 18 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[n-heptyloxymethyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of hepyt-2-yn-1-olbenzenesulfonate instead of MeI. MS (CI): m/z=448 (M+NH₄).

EXAMPLE 19 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[n-octyloxymethyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of oct-2-yn-1-olbenzenesulfonate instead of MeI. MS (CI): m/z=462 (M+NH₄).

EXAMPLE 20 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[n-nonyloxymethyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of non-2-yn-1-olbenzenesulfonate instead of MeI. MS (CI): m/z=476 (M+NH₄).

EXAMPLE 21 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[n-decyloxymethyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of dec-2-yn-1-olbenzenesulfonate instead of MeI. MS (CI): m/z=490 (M+NH₄).

EXAMPLE 22 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[3-methylbut-1-oxymethyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of1-iodo-3-methylbutane instead of MeI. MS (CI): m/z=403 (M+H).

EXAMPLE 23 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[2-propoxymethyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 9 was followed, with the use of 2-iodopropaneinstead of MeI. MS (CI): m/z=375 (M+H).

EXAMPLE 24 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[2-(tetrahydropyranyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To a solution of sordaricin benzyl ester (10 mg) in 3 mL of CH₂ Cl₂ wasadded 3,4-dihydro-2H-pyran (50 μL) and a catalytic amount of PPTS. Themixture was stirred at room temperature overnight. NEt₃ (1 mL) was addedand the mixture was concentrated in vacuo. After purification by PTLC,the benzyl ester of the title compound was obtained. To a MeOH solutionof this benzyl ester was added Pearlman's catalyst. The mixture wasstirred under hydrogen (balloon pressure) for 15 minutes. The mixturewas filtered through cotton. The filtrate was concentrated in vacuo togive the title compound. MS (CI): m/z=434 (M+NH₄).

EXAMPLE 25 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(1-ethoxyethoxy)methyl)]-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The same procedure for the preparation of the compound from Example 24was followed with the use of ethyl vinyl ether instead3,4-dihydro-2H-pyran. ¹ H NMR (CDCl₃): δ0.78-2.50 (28H, m), 3.20-4.20(4H, m), 4.60-4.80 (1H, m), 6.03-6.10 (1H), 9.84-9.86 (1H).

EXAMPLE 26 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(benzyloxy)methyl)]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylic acid

To a DMF solution of sordaricin p-methoxybenzyl ester was added excessbenzyl bromide and sodium hydride. The mixture was stirred at roomtemperature overnight. After aqueous work-up (ether) and purification byPTLC, the 4-methoxybenzyl ester of the title compound was obtained. Theester was then dissolved in excess formic acid. The mixture was stirredat room temperature for 3 hours. After concentration in vacuo andpurification by PTLC, the title compound was obtained. MS (CI): m/z=423(M+H).

EXAMPLE 27 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(4-bromobenzyloxy)methyl)]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 26 was followed, with the use of 4-bromobenzylbromide instead of benzyl bromide. MS (CI): m/z=501, 503 (M+H).

EXAMPLE 28 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(1-but-2-enyloxy)methyl)]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 26 was followed, with the use of crotylchloride instead of benzyl bromide. MS (CI): m/z=387 (M+H).

EXAMPLE 29 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(1-pent-2-enyloxy)methyl)]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 26 was followed, with the use oftrans-1-bromo-2-pentene instead of benzyl bromide. MS (CI): m/z=401(M+H).

EXAMPLE 30 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(isobutenyloxy)methyl)]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The procedure of Example 26 was followed, with the use of2-(bromomethyl)propene instead of benzyl bromide. MS (CI): m/z=387(M+H).

EXAMPLES 31-36

Following the procedure of Example 3, the following esters wereprepared:

    ______________________________________                                         ##STR13##                                                                                              Mass Spectrum                                       Acylating Agent                                                                            Product (R)  (m/z)                                               ______________________________________                                        CH.sub.3 (CH.sub.2).sub.2 COCl                                                             CH.sub.3 (CH.sub.2).sub.2 CO--                                                             420.4 ((M + NH.sub.4)                               CH.sub.3 (CH.sub.2).sub.3 COCl                                                             CH.sub.3 (CH.sub.2).sub.3 CO--                                                             434.4 (M + NH.sub.4)                                CH.sub.3 (CH.sub.2).sub.4 COCl                                                             CH.sub.3 (CH.sub.2).sub.4 CO--                                                             448.5 (M + NH.sub.4)                                CH.sub.3 (CH.sub.2).sub.6 COCl                                                             CH.sub.3 (CH.sub.2).sub.6 CO--                                                             476.5 (M + NH.sub.4)                                (CH.sub.3).sub.3 COCl                                                                      (CH.sub.3).sub.3 CCO--                                                                     434.3 (M + NH.sub.4)                                (CH.sub.3).sub.2 CHCH.sub.2 COCl                                                           (CH.sub.3).sub.2 CHCH.sub.2 CO--                                                           434.4 (M + NH.sub.4)                                ______________________________________                                    

EXAMPLES 37-46

Following the procedure of Example 3, the following esters may beprepared:

    ______________________________________                                         ##STR14##                                                                    Acylating Agent                                                                              Product (R)  MW                                                ______________________________________                                        (CH.sub.3).sub.2 CHCOCl                                                                      (CH.sub.3).sub.2 CHCO--                                                                    402.5296                                          C.sub.6 H.sub.5 COCl                                                                         C.sub.6 H.sub.5 CO--                                                                       436.5468                                          m-CH.sub.3 C.sub.6 H.sub.4 COCl                                                              m-CH.sub.3 C.sub.6 H.sub.4 CO--                                                            450.5736                                          o-FC.sub.6 H.sub.4 COCl                                                                      o-FC.sub.6 H.sub.4 CO--                                                                    454.5373                                          C.sub.6 H.sub.5 CH.sub.2 COCl                                                                C.sub.6 H.sub.5 CH.sub.2 CO--                                                              450.5736                                          C.sub.6 H.sub.5 (CH.sub.2).sub.2 COCl                                                        C.sub.6 H.sub.5 (CH.sub.2).sub.2 CO--                                                      464.6004                                          1-Naphthoyl Chloride                                                                         1-Naphthoyl  486.6066                                          2-Naphthoyl Chloride                                                                         2-Naphthoyl  486.6066                                          2-Pyrazinecarbonyl                                                                           2-Pyrazinecarbonyl                                                                         438.5224                                          Chloride                                                                      2-Furoyl Chloride                                                                            2-Furoyl     426.5084                                          ______________________________________                                    

EXAMPLE 47 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(2-pyridylcarboxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A solution of nicotinic acid (16 mg, 0.13 mmol) was prepared in 2 mL ofdry dichloromethane. N,N-Dimethylaminopyridine (2 mg, 0.01 mmol) wasadded followed by dicyclohexylcarbodiimide (27 mg, 0.13 mmol).Sordaricin benzyl ester (50 mg, 0.118 mmol) was added and the mixturewas stirred under an inert atmosphere for 24 h. The mixture was filteredand the organic layer was washed twice with water, twice with 5% aceticacid and again with water. The organic phase was dried over sodiumsulfate, filtered and concentrated in vacuo. Purification by PTLC(2:1/hexanes:ethyl acetate) gave 41.5 mg (67%) of the benzyl ester ofthe title compound.

The purified product was dissolved in 2 mL of methanol and approximately2 mg of palladium hydroxide on carbon was added. The reaction vessel wasflushed with hydrogen gas and stirred vigorously at room temperature forone hour. The catalyst was removed by filtration through a celite padand the filtrate was concentrated in vacuo to give the crude product.Purification by HPLC (C18 Zorbax, 40:60/95% water with 5%acetonitrile:95% acetonitrile with 5% water, 210 nm detection) gave 6.3mg (18%) of the title compound. Mass Spectrum (ESI): m/z=438.3 (M+H).

EXAMPLES 48-49 ##STR15##

Following the procedure of Example 47 but substituting the appropriatecarboxylic acid for nicotinic acid, the following compounds wereprepared.

    ______________________________________                                                                      Mass Spectrum                                   Carboxylic Acid                                                                             Product (R)     (m/z)                                           ______________________________________                                         ##STR16##                                                                                   ##STR17##      452.4 (M + H)                                    ##STR18##                                                                                   ##STR19##      488.4 (M + H)                                   ______________________________________                                    

EXAMPLES 50-52

To a CH₂ Cl₂ solution of sordaricin p-methoxybenzyl ester (0.5 mg) isadded NEt₃ (0.2 mL), followed by the appropriate acid chloride (10-100mg). A catalytic amount of DMAP is added. The mixture is stirred at roomtemperature overnight. After purification by PTLC, the p-methoxybenzylester of the title compound is obtained. The ester is then dissolved inexcess formic acid. The mixture is stirred at room temperature for 3hours or a time sufficient to remove the protecting group. Afterconcentration in vacuo and purification by PTLC, the title compound isobtained.

    ______________________________________                                         ##STR20##                                                                    Acylating Agent                                                                              Product (R)   MW                                               ______________________________________                                        CH.sub.2 ═CH(CH.sub.2).sub.2 COCl                                                        CH.sub.2 ═CH(CH.sub.2).sub.2 CO--                                                       414.5406                                         (CH.sub.3).sub.2 C═CHCOCl                                                                (CH.sub.3).sub.2 C═CHCO                                                                 414.5406                                         p-ClC.sub.6 H.sub.4 COCl                                                                     p-ClC.sub.6 H.sub.4 CO--                                                                    470.9919                                         ______________________________________                                    

EXAMPLES 53-54

p-Methoxybenzylsordaricin ester was dissolved in dichloromethane andplaced under an inert atmosphere of dry nitrogen. A slight molar excessof diisopropylethylamine was added and the mixture was cooled in a dryice-acetone bath. A slight molar excess of acylating agent as describedbelow was added and the mixture was stirred at -78° C. until asignificant portion of the starting alcohol was consumed. The reactionwas quenched, warmed to room temperature and concentrated in vacuo.Purification by PTLC gave the p-methoxybenzyl ester of the desiredcompound.

The compound obtained above was dissolved in formic acid and stirred atroom temperature for 3 hours or a time sufficient to remove thep-methoxybenzyl ester. After concentration in vacuo and purification byPTLC, the desired compound as shown below was obtained.

    ______________________________________                                         ##STR21##                                                                                               Mass Spectrum                                      Acylating Agent                                                                             Product (R)  (m/z)                                              ______________________________________                                        H.sub.2 C═CHCOCl                                                                        H.sub.2 C═CHCO--                                                                       404 (M + NH.sub.4)                                 H.sub.3 CCH═CHCOCl                                                                      H.sub.3 CCH═CHCO--                                                                     418 (M + NH.sub.4)                                 ______________________________________                                    

EXAMPLES 55-67

    ______________________________________                                         ##STR22##                                                                    Acylating Agent                                                                              Product (R)    MW                                              ______________________________________                                        cyclohexane carboxylic                                                                       cyclohexane carbonyl                                                                         442.5942                                        acid                                                                          cyclopentane carboxylic                                                                      cyclopentane carbonyl                                                                        428.5674                                        acid                                                                          cyclobutane carboxylic                                                                       cyclobutane carbonyl                                                                         414.5406                                        acid                                                                          p-CH.sub.3 C.sub.6 H.sub.4 CO.sub.2 H                                                        p-CH.sub.3 C.sub.6 H.sub.4 CO--                                                              450.5736                                        m-FC.sub.6 H.sub.4 CO.sub.2 H                                                                m-FC.sub.6 H.sub.4 CO--                                                                      454.5373                                        4-biphenylcarboxylic                                                                         4-biphenylcarbonyl                                                                           512.6444                                        acid                                                                          3-biphenylcarboxylic                                                                         3-biphenylcarbonyl                                                                           512.6444                                        acid                                                                          4,4'-terphenylcarboxylic                                                                     4,4'-terphenylcarbonyl                                                                       588.7420                                        acid                                                                          9-anthracenecarboxylic                                                                       9-anthracenecarbonyl                                                                         536.6664                                        acid                                                                          2-pyrrolecarboxylic acid                                                                     2-pyrrolecarbonyl                                                                            425.5236                                        (CH.sub.3).sub.2 CH(CH.sub.2).sub.2 CO.sub.2 H                                               (CH.sub.3).sub.2 CH(CH.sub.2).sub.2 CO--                                                     430.5832                                        HCO.sub.2 H    HCO--          360.4492                                        2-thiophenecarboxylic                                                                        2-thiophenecarbonyl                                                                          442.5690                                        acid                                                                          ______________________________________                                    

EXAMPLES 68-70

To a THF solution of the acid (0.16 mmol) is added NEt₃ (34 μL),followed by 2,4,6-trichlorobenzoyl chloride (25 μL). The 5 mixture isstirred at room temperature for 15 minutes. Sordarin p-methoxybenzylester (1.0 mg) in 1 mL of THF is then added to this mixture, followed bythe addition of DMAP (20 mg). This mixture is stirred at roomtemperature for 1 hour. After purification by PTLC, the p-methoxybenzylester of the desired compound is obtained. The ester is then dissolvedin excess formic acid. The mixture is stirred at room temperature for 3hours or a time sufficient to remove the protecting group. Afterconcentration in vacuo and purification by PTLC, the desired compound isobtained.

    ______________________________________                                         ##STR23##                                                                    Acylating Agent                                                                              Product (R)  MW                                                ______________________________________                                        o-ClC.sub.6 H.sub.4 CO.sub.2 H                                                               o-ClC.sub.6 H.sub.4 CO--                                                                   470.9919                                          3,5-dibromobenzoic                                                                           3,5-dibromobenzoyl                                                                         594.3390                                          acid                                                                          cyclopropane   cyclopropylcarbonyl                                                                        400.5138                                          carboxylic acid                                                               ______________________________________                                    

EXAMPLES 71-81

In a manner analogous to that in Example 4, the following carbonates maybe prepared:

    ______________________________________                                         ##STR24##                                                                    Acylating Agent                                                                              Product (R)    MW                                              ______________________________________                                        ethyl chloroformate                                                                          ethoxycarbonyl 404.5022                                        phenyl chloroformate                                                                         phenoxycarbonyl                                                                              452.5462                                        tert-butyl chloroformate                                                                     tert-butoxycarbonyl                                                                          432.5558                                        cyclopropyl    cyclopropoxycarbonyl                                                                         416.5132                                        chloroformate                                                                 n-butyl chloroformate                                                                        n-butoxycarbonyl                                                                             432.5558                                        (CH.sub.3).sub.2 CHCH.sub.2 OCOCl                                                            (CH.sub.3).sub.2 CHCH.sub.2 OCO--                                                            432.5558                                        sec-butyl chloroformate                                                                      sec-butoxycarbonyl                                                                           432.5558                                        isopropyl chloroformate                                                                      isopropoxycarbonyl                                                                           418.5290                                        C.sub.6 H.sub.5 (CH.sub.2).sub.2 OCOCl                                                       C.sub.6 H.sub.5 (CH.sub.2).sub.2 OCO--                                                       480.5998                                        n-propyl chloroformate                                                                       n-propoxycarbonyl                                                                            418.5290                                        cyclohexylchloroformate                                                                      cyclohexoxycarbonyl                                                                          458.5936                                        ______________________________________                                    

EXAMPLES 82-86

To a CH₂ Cl₂ solution of sordaricin p-methoxybenzyl ester (0.5 mg) isadded NEt₃ (0.2 mL), followed by the appropriate acid chloride (10-100mg). A catalytic amount of DMAP is added. The mixture is stirred at roomtemperature overnight. After purification by PTLC, the p-methoxybenzylester of the title compound is obtained. The ester is then dissolved inexcess formic acid. The mixture is stirred at room temperature for0.25-3 hours or a time sufficient to remove the protecting group. Afterconcentration in vacuo and purification by PTLC, the title compound isobtained.

    ______________________________________                                         ##STR25##                                                                    Acylating Agent                                                                             Product (R)     MW                                              ______________________________________                                        benzyl chloroformate                                                                        benzyloxycarbonyl                                                                             466.5730                                        p-methylbenzyl                                                                              p-              480.5998                                        chloroformate methylbenzyloxycarbonyl                                         1-naphthyl    1-naphthyloxycarbonyl                                                                         516.6328                                        chloroformate                                                                 2-naphthalene 2-naphthoxycarbonyl                                                                           502.6060                                        chloroformate                                                                 CH.sub.2 ═CH(CH.sub.2).sub.2 OCOCl                                                      CH.sub.2 ═CH(CH.sub.2).sub.2 OCO--                                                        430.5400                                        ______________________________________                                    

EXAMPLES 87-88

Following the procedure of that in Examples 4, the following carbonatesmay be prepared:

    ______________________________________                                         ##STR26##                                                                    Acylating Agent                                                                              Product (R)    MW                                              ______________________________________                                        allyl chloroformate                                                                          allyloxycarbonyl                                                                             416.5132                                        crotyl chloroformate                                                                         crotyloxycarbonyl                                                                            430.5400                                        ______________________________________                                    

EXAMPLES 89-103

In a manner analogous to that in Example 5, the following carbamates maybe prepared:

    ______________________________________                                         ##STR27##                                                                    Acylating Agent                                                                              Product (R)      MW                                            ______________________________________                                        methyl isocyanate                                                                            methylaminocarbonyl                                                                            389.4906                                      phenyl isocyanate                                                                            anilinocarbonyl  451.5614                                      tert-butyl isocyanate                                                                        tert-butylaminocarbonyl                                                                        431.5710                                      cyclobutyl isocyanate                                                                        cyclobutylaminocarbonyl                                                                        429.5552                                      (CH.sub.3).sub.2 CHCH.sub.2 NCO                                                              (CH.sub.3).sub.2 CHCH.sub.2 NHCO--                                                             431.5710                                      p-CH.sub.3 C.sub.6 H.sub.4 NCO                                                               p-CH.sub.3 C.sub.6 H.sub.4 NHCO                                                                465.5882                                      isopropyl isocyanate                                                                         isopropylaminocarbonyl                                                                         417.5442                                      cyclopentyl isocyanate                                                                       cyclopentylaminocarbonyl                                                                       443.5820                                      naphthalene-1- 1-naphthylaminocarbonyl                                                                        501.6212                                      isocyanate                                                                    dimethylaminocarbonyl                                                                        dimethylaminocarbonyl                                                                          403.5174                                      chloride                                                                      N-methyl-N-    N-methyl-N-      445.5978                                      butylaminocabonyl                                                                            butylaminocabonyl                                              chloride                                                                      N-ethyl-N-     N-ethyl-N-       403.5174                                      benzylaminocarbonyl                                                                          benzylaminocarbonyl                                            chloride                                                                      N-phenyl-N-2-  N-phenyl-N-2-    577.7188                                      naphthylaminocarbonyl                                                                        naphthylaminocarbonyl                                          chloride                                                                      N-methyl-N-    N-methyl-N-      465.5882                                      phenylaminocarbonyl                                                                          phenylaminocarbonyl                                            chloride                                                                      N-methyl-N-    N-methyl-N-      429.5552                                      cyclopropylaminocarbonyl                                                                     cyclopropylaminocarbonyl                                       chloride                                                                      ______________________________________                                    

EXAMPLES 104-107

To a CHCl₃ solution of sordaricin p-methoxybenzyl ester (0.5 mg) isadded the appropriate isocyanate (10-100 mg) and a catalytic amount ofDMAP. The mixture is refluxed for 4 hours or until the reaction nolonger proceeds. After concentration in vacuo and purification by PTLC,the p-methoxybenzyl ester of the desired compound is obtained. The esteris then dissolved in excess formic acid. The mixture is stirred at roomtemperature for 3 hours or a time sufficient to remove the protectinggroup. After concentration in vacuo and purification by PTLC, thedesired compound is obtained.

    ______________________________________                                         ##STR28##                                                                    Acylating Agent                                                                              Product (R)    MW                                              ______________________________________                                        benzyl isocyanate                                                                            benzylaminocarbonyl                                                                          465.5882                                        allyl isocyanate                                                                             allylaminocarbonyl                                                                           415.5284                                        o-iodophenyl   o-iodoanalinocarbonyl                                                                        577.4580                                        isocyanate                                                                    dibenzylaminocarbonyl                                                                        dibenzylaminocarbonyl                                                                        555.7126                                        chloride                                                                      ______________________________________                                    

EXAMPLE 108 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[((Z)-but-2-enyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Utilizing a procedure similar to that as described in Example 26, butusing (Z)-1-p-toluenesulfonyloxy-2-butene in the place of benzylbromide, the title compound was prepared. Partial ¹ H NMR (CDCl₃): δ0.82(3H, d), 0.98 (3H, d), 1.03 (3H, d), 3.24 (1H, d), 3.98 (1H, d), 4.04(2H, d), 5.50 (1H, m), 5.76 (1H, m), 6.02 (1H, m), 9.89 (1H, s).

EXAMPLES 109-112

Following the procedure of Example 26, the following ethers wereprepared:

    ______________________________________                                         ##STR29##                                                                                                  Mass Spectrum                                   Alkylating Agent                                                                            Product (R-)    (m/z)                                           ______________________________________                                        1-p-toluenesulfonyloxy-2-                                                                   H.sub.3 CC.tbd.CCH.sub.2 --                                                                   402.4                                           butyne                        (M + NH.sub.4)                                  1-chloro-2-pentyne                                                                          CH.sub.3 CH.sub.2 C.tbd.CCH.sub.2 --                                                          416.4                                                                         (M + NH.sub.4)                                  (Z)-1-p-toluenesulfonyloxy-  2-pentane                                                       ##STR30##      40.41(M + H)                                    (Z)-1-p-toluenesulfonyloxy-  2-hexene                                                        ##STR31##      415.4(M + H)                                    ______________________________________                                    

EXAMPLES 113-115

Following the procedure of Example 26, the following ethers may beprepared:

    ______________________________________                                         ##STR32##                                                                    Alkylating Agent                                                                            Product (R-)    MW                                              ______________________________________                                        1-bromo-2-propyne                                                                           HC.tbd.CCH.sub.2 --                                                                           370.4876                                        1-iodo-5-hexyne                                                                             HC.tbd.CCH.sub.2 CH.sub.2 CH.sub.2 CH.sub.2 --                                                412.5680                                        cis-1-trimethylsilyloxy-                                                                    cis-4-hydroxy-2-butenyl                                                                       402.5296                                        4-bromo-2-butene                                                              ______________________________________                                    

EXAMPLES 116-133

Following the procedure of Example 26, but substituting the appropriatealkylating agent for benzyl bromide, the following ethers were prepared:

    ______________________________________                                         ##STR33##                                                                                                 Mass Spectrum                                    Alkylating Agent                                                                           Product (R-)    (m/z)                                            ______________________________________                                        3-methylbenzyl                                                                             3-methylbenzyl  437 (M + H)                                      bromide                                                                       4-methylbenzyl                                                                             4-methylbenzyl  437 (M + H)                                      bromide                                                                       2-methylbenzyl                                                                             2-methylbenzyl  437 (M + H)                                      chloride                                                                      2-fluorobenzyl                                                                             2-fluorobenzyl  441 (M + H)                                      bromide                                                                       3-fluorobenzyl                                                                             3-fluorobenzyl  441 (M + H)                                      bromide                                                                       4-fluorobenzyl                                                                             4-fluorobenzyl  441 (M + H)                                      bromide                                                                       3-methoxybenzyl                                                                            3-methoxybenzyl 453 (M + H)                                      bromide                                                                       1-naphthyl bromide                                                                         1-naphthyl      473 (M + H)                                      2-phenylbenzyl                                                                             2-phenylbenzyl  499 (M + H)                                      bromide                                                                       2,3-difluorobenzyl                                                                         2,3-difluorobenzyl                                                                            459 (M + H)                                      bromide                                                                       2,4-difluorobenzyl                                                                         2,4-difluorobenzyl                                                                            459 (M + H)                                      bromide                                                                       2,5-difluorobenzyl                                                                         2,5-difluorobenzyl                                                                            459 (M + H)                                      bromide                                                                       2,6-difluorobenzyl                                                                         2,6-difluorobenzyl                                                                            459 (M + H)                                      bromide                                                                       3,5-difluorobenzyl                                                                         3,5-difluorobenzyl                                                                            459 (M + H)                                      bromide                                                                       3-carbomethoxybenzyl                                                                       3-carbomethoxybenzyl                                                                          481 (M + H)                                      bromide                                                                       1-(benzene-  (3-cyclohexenyl)methyl                                                                        427 (M + H)                                      sulfonyloxymethyl)-3-                                                         cyclohexene                                                                   5-chloro-2-(chloro-                                                                        (5-chloro-2-    480 (M + NH.sub.4)                               methyl)thiophene                                                                           thienyl)methyl                                                   2,5-dimethyl-3-                                                                            (2,5-dimethyl-3 442 (M + H)                                      (chloromethyl)isoxazole                                                                    isoxazolyl)methyl                                                ______________________________________                                    

EXAMPLE 134 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(2-hydroxyethyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-5methano-s-indacene-3a(1H)-carboxylic acid

A solution of sordaricin benzyl ester (735.3 mg, 1.74 mmol) and1-benzyloxy-2-benzenesulfonyloxyethane (3.94 g, 13.5 mmol) was preparedin 20 mL of N,N-dimethylformamide. Sodium hydride (210 mg of a 60%dispersion in mineral oil, 5.25 mmol) was added at room temperature andthe mixture was stirred overnight. The reaction was quenched carefullywith water and extracted with ether. Purification by flashchromatography gave 756 mg (78%) of benzyl[1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)] 8a-[(2-benzyloxyethyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicester.

The product from above (700 mg, 1.26 mmol) was dissolved in 15 mL ofmethanol and 200 mg of palladium hydroxide on carbon (Pearlman'scatalyst) was added. The mixture was stirred under one atmosphere ofhydrogen gas for 30 minutes. The catalyst was removed by filtration andthe filtrate was concentrated under reduced pressure to give the titlecompound. Mass spectrum (m/z): 377 (M+H).

EXAMPLE 135 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(3-hydroxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

A solution of sordaricin benzyl ester (695 mg, 1.64 mmol) and1-benzyloxy-3-benzenesulfonyloxypropane (5.04 g, 16.5 mmol) was preparedin 20 mL of N,N-dimethylformarnide. Sodium hydride (200 mg of a 60%dispersion in mineralqoil, 5.00 mmol) was added at room temperature andthe mixture was stirred overnight. The reaction was quenched carefullywith water and extracted with ether. Purification by flashchromatography gave 704 mg (75%) of benzyl[1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(3-benzyloxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicester. ¹ H NMR (CDCl₃): δ.

The product from above (308.5 mg, 0.541 mmol) was dissolved in 10 mL ofmethanol and 100 mg of palladium hydroxide on carbon (Pearlman'scatalyst) was added. The mixture was stirred under one atmosphere ofhydrogen gas for 30 minutes. The catalyst was removed by filtration andthe filtrate was concentrated under reduced pressure to give 211 mg(quantitative yield) of the title compound. Mass spectrum (m/z): 391(M+H).

EXAMPLE 136 Benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(2-hydroxyethyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate

The product from Example 134 was dissolved in 15 mL of dryN,N-dimethylformamide containing 1 g of sodium hydrogen carbonate and1.5 mL of benzyl bromide. The mixture was stirred overnight andpartitioned between water and ether. The organic phase was dried overanhydrous sodium sulfate and purified by flash chromatography to give534 mg (91%) of the title compound.

EXAMPLE 137 Benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(3-hydroxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate

The product from Example 135 was dissolved in 10 mL of dryN,N-dimethylformamide and 1 g of sodium hydrogen carbonate and 1 mL ofbenzyl bromide were added. The mixture was stirred overnight at roomtemperature and partitioned between water and ether. The organic phasewas dried over anhydrous sodium sulfate and purified by flashchromatography to give 233.7 mg (90%) of the title compound.

EXAMPLES 138-140 Benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(2-hydroxyethoxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylatewas dissolved in dry N,N-dimethylformamide and excess sodium hydride(60% dispersion in mineral oil) and excess alkylating agent as indicatedbelow were added. The mixture was stirred until most of the startingalcohol compound was consumed as determined by thin layerchromatography. The reaction mixture was quenched carefully with waterand partitioned between ether and water. The organic phase was driedover anhydrous sodium sulfate,filtered, the volatiles removed underreduced pressure and the residue purified by PTLC to obtain the desiredbenzyl ester product.

The benzyl ester product from above was dissolved in methanol andpalladium hydroxide on carbon was added. The reaction mixture wasstirred vigorously under one atmosphere of hydrogen for 30 minutes oruntil thin layer chromatography indicated the reaction to be complete.The catalyst was removed by filtration and the filtrate concentratedunder reduced pressure to give the indicated product.

    ______________________________________                                         ##STR34##                                                                                               Mass Spectrum                                      Alkylating Agent                                                                            Product (R-) (m/z)                                              ______________________________________                                        iodoethane    C.sub.2 H.sub.5 O(CH.sub.2).sub.2 --                                                       405 (M + H)                                        n-butyl iodide                                                                              n-C.sub.4 H.sub.9 O(CH.sub.2).sub.2 --                                                     433 (M + H)                                        isobutenyl bromide                                                                          i-C.sub.4 H.sub.9 O(CH.sub.2).sub.2 --                                                     433 (M + H)                                        ______________________________________                                    

EXAMPLES 141-146 Benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(3-hydroxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylatewas dissolved in dry N,N-dimethylformamide and excess sodium hydride(60% dispersion in mineral oil) and excess alkylating agent as indicatedbelow were added. The mixture was stirred until most of the startingalcohol compound was consumed as determined by thin layerchromatography. The reaction mixture was quenched carefully with waterand partitioned between ether and water. The organic phase was driedover anhydrous sodium sulfate, filtered, the volatiles removed underreduced pressure and the residue purified by PTLC to obtain the desiredbenzyl ester product.

The benzyl ester product from above was dissolved in methanol andpalladium hydroxide on carbon was added. The reaction mixture wasstirred vigorously under one atmosphere of hydrogen for 30 minutes oruntil thin layer chromatography indicated the reaction to be complete.The catalyst was removed by filtration and the filtrate concentratedunder reduced pressure to give the indicated product.

    ______________________________________                                         ##STR35##                                                                                                 Mass Spectrum                                    Alkylating Agent                                                                            Product (R-)   (m/z)                                            ______________________________________                                        iodomethane   CH.sub.3 O(CH.sub.2).sub.3 --                                                                405 (M + H)                                      iodoethane    C.sub.2 H.sub.5 O(CH.sub.2).sub.3 --                                                         419 (M + H)                                      allyl bromide n-C.sub.3 H.sub.7 O(CH.sub.2).sub.3 --                                                       433 (M + H)                                      n-butyl iodide                                                                              n-C.sub.4 H.sub.9 O(CH.sub.2).sub.3 --                                                       447 (M + H)                                      n-pentyl iodide                                                                             n-C.sub.5 H.sub.11 O(CH.sub.2).sub.3 --                                                      461 (M + H)                                      1-benzyloxy-3-                                                                              HO(CH.sub.2).sub.3 O(CH.sub.2).sub.3 --                                                      466 (M + NH.sub.4)                               benzenesulfonyloxypropane                                                     ______________________________________                                    

EXAMPLE 147 Benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(3-hydroxypropyl)-3-oxypropyloxymethyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylat

The product of Example 146 was dissolved in dry N,N-dimethylformamideand an excess of sodium hydrogen carbonate and an excess of benzylbromide were added. The mixture was stirred overnight at roomtemperature and partitioned between water and ether. The organic phasewas dried over anhydrous sodium sulfate and purified by flashchromatography to give the title compound.

EXAMPLES 148-152 Benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(3-hydroxypropyl)-3-oxypropyloxymethyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylatewas dissolved in dry N,N-dimethylformamide and excess sodium hydride(60% dispersion in mineral oil) and excess alkylating agent as indicatedbelow were added. The mixture was stirred until most of the startingalcohol compound was consumed as determined by thin layerchromatography. The reaction mixture was quenched carefully with waterand partitioned between ether and water. The organic phase was driedover anhydrous sodium sulfate, filtered, the volatiles removed underreduced pressure and the residue purified by PTLC to obtain the desiredbenzyl ester product.

The benzyl ester product from above was dissolved in methanol andpalladium hydroxide on carbon was added. The reaction mixture wasstirred vigorously under one atmosphere of hydrogen for 30 minutes oruntil thin layer chromatography indicated the reaction to be complete.The catalyst was removed by filtration and the filtrate concentratedunder reduced pressure to give the indicated product.

    ______________________________________                                         ##STR36##                                                                                                Mass Spectrum                                     Alkylating Agent                                                                         Product (R-)     (m/z)                                             ______________________________________                                        iodomethane                                                                              CH.sub.3 O(CH.sub.2).sub.3 O(CH.sub.2).sub.3 --                                                480 (M + NH.sub.4)                                iodoethane C.sub.2 H.sub.5 O(CH.sub.2).sub.3 O(CH.sub.2).sub.3 --                                         494 (M + NH.sub.4)                                allyl bromide                                                                            n-C.sub.3 H.sub.7 O(CH.sub.2).sub.3 O(CH.sub.2).sub.3 --                                       508 (M + NH.sub.4)                                n-butyl iodide                                                                           n-C.sub.4 H.sub.9 O(CH.sub.2).sub.3 O(CH.sub.2).sub.3 --                                       522 (M + NH.sub.4)                                n-pentyl iodide                                                                          n-C.sub.5 H.sub.11 O(CH.sub.2).sub.3 O(CH.sub.2).sub.3                                         536 (M + NH.sub.4)                                ______________________________________                                    

EXAMPLE 153 Benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(R-2-hydroxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate

Sordaricin benzyl ester (450 mg, 1.07 mmol) and the benzenesulfonate of(S)-glycidol (approximately 3 g) were dissolved in 20 mL of dryN,N-dimethylformamide. Sodium hydride (128 mg of a 60% dispersion inmineral oil, 3.2 mmol) was added and the mixture was stirred at roomtemperature overnight. The reaction was caefully quenched with water andextracted with ether. The ether layer was dried with anhydrous sodiumsulfate, filtered and concentrated under reduced pressure. Purificationof the residue by flash chromatography gave 351.7 mg (69%) of benzyl[1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(S-2,3-epoxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate.

The product from above (340 mg, 0.711 mmol) was dissolved in 10 mL ofmethanol and 100 mg of palladium hydroxide on carbon (Pearlman'scatalyst) was added. The mixture was stirred vigorously under oneatmosphere of hydrogen for 30 minutes. The catalyst was removed byfiltration and the filtrate was concentrated under reduced pressure.

The residue was dissolved in 15 mL of N,N-dimethylformamide and excesssolid sodium hydrogen carbonate (0.5 g) and benzyl bromide (2 mL) wereadded. The mixture was stirred overnight and partitioned between etherand water. The organic phase was dried over anhydrous sodium sulfate,filtered and concentrated in vacuo. The residue was purified by flashchromatography to give 205 mg (61%) of the title compound. ¹ H NMR(CDCl₃): δ0.51 (3H, d, J=7.1), 0.80 (3H, d, J=6.7), 1.00 (3H, d, J=6.8),1.09 (3H, d, J=6.4), 0.80-2.30 (12H, m), 2.73 (1H, t, J=3.9), 3.08 (1H,dd, J=8.2, 9.6), 3.28 (1H, dd, J=3.0, 9.7), 3.47 (1H, d, J=8.9), 3.61(1H, d, J=8.9), 3.88 (1H, m), 5.13 (1H, d, J=11.9), 5.22 (1H, d,J=11.9), 6.03 (1H, dd, J=1.3, 3.4), 7.28-7.38 (5H, m), 9.66 (1H, s).

EXAMPLES 154-158 Benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(R-2-hydroxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylatewas dissolved in dry N,N-dimethylforrnamide and excess sodium hydride(60% dispersion in mineral oil) and excess alkylating agent as indicatedbelow were added. The mixture was stirred until most of the startingalcohol compound was consumed as determined by thin layerchromatography. The reaction mixture was quenched carefully with waterand partitioned between ether and water. The organic phase was driedover anhydrous sodium sulfate, filtered, the volatiles removed underreduced pressure and the residue purified by PTLC to obtain the desiredbenzyl ester product.

The benzyl ester product from above was dissolved in methanol andpalladium hydroxide on carbon was added. The reaction mixture wasstirred vigorously under one atmosphere of hydrogen for 30 minutes oruntil thin layer chromatography indicated the reaction to be complete.The catalyst was removed by filtration and the filtrate concentratedunder reduced pressure to give the indicated product.

    ______________________________________                                         ##STR37##                                                                                             Mass Spectrum                                        Alkylating Agent                                                                             Product (R-)                                                                            (m/z)                                                ______________________________________                                        iodomethane    CH.sub.3 --                                                                             405 (M + H)                                          iodoethane     C.sub.2 H.sub.5 --                                                                      419 (M + H)                                          allyl bromide  n-C.sub.3 H.sub.7 --                                                                    450 (M + NH.sub.4)                                   n-butyl iodide n-C.sub.4 H.sub.9 --                                                                    464 (M + NH.sub.4)                                   n-pentyl iodide                                                                              n-C.sub.5 H.sub.11 --                                                                   478 (M + NH.sub.4)                                   ______________________________________                                    

EXAMPLE 159 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(m-carboxybenzyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The product of Example 130 was dissolved in methanol and aqueous sodiumhydroxide was added and the mixture was stirred until starting materialwas consumed as determined by thin layer chromatographic analysis. Thereaction was acidified and extracted with ethyl acetate to give thetitle compound. Mass spectrum (m/z): 467 (M+H).

EXAMPLE 160 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(R-2-aminopropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

The compound of Example 16 was dissolved in N,N-dimethylformamide andexcess benzyl bromide was added followed by excess solid sodium hydrogencarbonate. The reaction was stirred overnight. The mixture wasconcentrated in vacuo, chloroform was added and the mixture wasfiltered. Concentration under reduced pressure followed by purificationof the residue by PTLC gave benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(R-2-hydroxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate.

The benzyl ester from above was dissolved in dichloromethane andtriethylamine was added followed by methanesulfonyl chloride. Thereaction mixture was stirred for 30 minutes, concentrated in vacuo andthe residue purified by PTLC to give benzyl[1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(R-2-methanesulfonyloxypropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate.

The methanesulfonate obtained above was dissolved in toluene and excesstetrabutylammonium azide in toluene was added. The mixture was heated to70° C. until the starting material was consumed. After concentration invacuo and purification by PTLC benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(S-2-azidopropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylatewas obtained.

Benzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(S-2-azidopropyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate was dissolved in methanol and palladium hydroxide oncarbon was added. The mixture was stirred vigorously under oneatmosphere of hydrogen for 30 minutes. The catalyst was removed byfiltration and the filtrate was concentrated under reduced pressure togive the title compound. ¹ H NMR (CD₃ OD): δ0.82 (3H, m), 0.91 (3H, m),0.93 (3H, m), 1.10 (3H, m), 1.10-2.40 (13H, m), 3.20-4.00 (5H, m), 5.87(1H, m), 9.83 (1H, s).

EXAMPLE 161 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(aminoacetyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Sordaricin benzyl ester and triethylamine were dissolved indichloromethane and cooled to -78° C. Chloroacetyl chloride andN,N-dimethylaminopyridine were added and the mixture was stirred at -78°C. for 3 hours. The mixture was warmed to room temperature and afterworkup was purified by PTLC to give benzyl[1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(chloroacetyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate.

The product from above was dissolved in toluene and excesstetrabutylammonium azide was added. The mixture was stirred overnightthen concentrated in vacuo and the residue purified by PTLC to givebenzyl [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(azidoacetyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate.

The azide obtained above was dissolved in methanol and palladiumhydroxide on carbon was added. The reaction was stirred under oneatmosphere of hydrogen for a time sufficient to remove the benzyl estergroup and reduce the azide. Concentration under reduced pressure gavethe title compound. ¹ H NMR (CD₃ OD): δ0.77 (3H, d, J=6.4), 0.93 (3H, d,J=6.7), 0.94 (3H, d, J=6.6), 1.50-2.30 (11H, m), 2.36 (1H, m), 2.51 (1H,m), 3.60-4.60 (4H, m), 5.90 (1H, s), 9.81 (1H, s).

EXAMPLE 162 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(4-hydroxybutyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

Sordaricin benzyl ester was dissolved in N,N-dimethylformamide followedby excess 1-bromo-4-tert-butyldimethylsilyloxybut-2-yne. An excessamount of sodium hydride (60% dispersion in mineral oil) was added andthe mixture was stirred at room temperature overnight. The mixture wasconcentrated in vacuo and the residue purified by PTLC to give benzyl[1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(4-tert-butyldimethylsilyloxybut-2-yn-1-yloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate.

The product above was dissolved in dry tetrahydrofuran and an excess oftetrabutylammonium fluoride (1M in tetrahydrofuran) was added. Themixture was stirred for one hour after which time it was concentrated invacuo. The residue was purified by PTLC to give benzyl[1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(4-hydroxybut-2-yn-1-yl-oxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylate.

The alcohol obtained above was dissolved in methanol and palladiumhydroxide on carbon (Pearlman's catalyst) was added. The reactionmixture was stirred under one atmosphere of hydrogen for 3 hours. Themixture was filtered and the filtrate was concentrated in vacuo to givethe title compound. ¹ H NMR (CDCl₃): δ0.82 (3H, d, J=6.9), 0.95 (3H, d,J=6.7), 1.02 (3H, d, J=6.8), 1.20-2.10 (15H, m), 2.33 (1H, m), 2.37 (1H,m), 3.26-4.00 (6H, m), 6.02 (1H, m), 9.88 (1H, s).

EXAMPLE 163 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(2-aminoethoxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

In a manner analogous to that of Example 160 but using the compound ofExample 134 in place of the compound of Example 16, the title compoundwas obtained. Mass spectrum (m/z): 376 (M+H).

EXAMPLE 164 [1R-(1α,3aβ,4β,4aβ,7β,7aα,8aβ)]8a-[(cyclopentylmethyloxy)methyl]-4-formyl-4,4a,5,6,7,7a,8,8a-octahydro-7-methyl-3-(1-methylethyl)-1,4-methano-s-indacene-3a(1H)-carboxylicacid

To an N,N-dimethylformamide (DMF) solution of sordaricin benzyl esterwas added excess cyclopentylmethyl benzenesulfonate, followed by NaH(60% oil dispersion). The mixture was stirred at room temperatureovernight. After concentration in vacuo and purification by PTLC, thebenzyl ester of the title compound was obtained. This derivative wasdissolved in methanol and palladium hydroxide on carbon (Pearlman'scatalyst) was added. Ths mixture was stirred under hydrogen (balloonpressure) until removal of the benzyl group was complete as determinedby TLC. After concentration in vacuo and purification by PTLC, the titlecompound was obtained. Mass spectrum (m/z): 415 (M+H).

What is claimed is:
 1. A compound having the formula I: ##STR38##wherein R is(a) C(═O)OR¹, (b) C(═O)NR² R³, (c) C(═O)R⁴, (d) CH(R²)OR⁵,(e) C(R⁶)(R⁷)(R⁸), ##STR39## with the proviso that when R¹² is CHO, R isnot (g); R¹ is (a) C₁ -C₁₄ alkyl, (b) C₂ -C₁₄ alkenyl, (c) C₂ -C₁₄alkynyl, (d) C₃ -C₂₀ cycloalkyl, (e) C₃ -C₂₀ cycloalkenyl, (f) aryl or(g) aryl C₁₋₆ alkyl; R² and R³ are independently(a) H or (b) R¹ ; R⁴is(a) H, (b) R¹ or (c) --(CH₂)_(m) NR² R³ ; R⁵ is(a) R¹ or (b)--(CH₂)_(x) O(CH₂)_(y) H; R⁶ is(a) H, (b) R¹, (c) --(CH₂)_(y) CHR₁₁(CH₂)_(z) H, (d) --(CH₂)_(y) C.tbd.--C(CH₂)_(z) H, (e) --(CH₂)_(y)C(R⁷)═CH(CH₂)_(z) H, (f) --(CH₂)_(y) C.tbd.C(CH₂)_(m) R¹¹, (g)--(CH₂)_(y) C(R⁷)═CH(CH₂)_(m) R¹¹, R⁷ and R⁸ are independently(a) H, or(b) C₁ -C₁₄ alkyl; R⁹ and R10 are independently(a) H, (a) C₁ -C₁₄ alkyl,(a) C₂ -C₁₄ alkenyl, (a) aryl C₁₋₆ alkyl; R¹¹ is(a) OR⁷, (b) OCH₂ CH₂(CH₂)_(n) OR⁷, or (c) NR² R³ ; R¹² is(a) --C(═O)R¹⁴, (b) --CH═NOH, or(c) --CH₂ OCH₃ ; R¹³ is(a) H, (b) --CH₂ C₆ H₅, (c) --CH₂ CH═CH₂,##STR40## R¹⁴ is (a) H, (b) C₁ -C₄ alkyl, (c) --CCl₃, (d) --CBr₃, (e)--CF₃, or (f) OH; n is 0 or 1; m is 1-6; x is 2-6; y is 0-6; z is 0-6 ora pharmaceutically or agriculturally acceptable salt thereof.
 2. Acompound of claim 1 whereinR¹² is --CHO; R is(a) C(═O)OR¹, (b) C(═O)NR²R³, (c) C(═O)R⁴, (d) CH₂ OR⁵, (e) C(R⁶)(R⁷)(R⁸), ##STR41##
 3. A compoundof claim 1 wherein R¹² is --CHOR¹³ is H, R is C(═O)OR¹.
 4. A compound ofclaim 1 whereinR¹² is --CHO R¹³ is H, R is C(═O)NR² R³.
 5. A compound ofclaim 1 whereinR¹² is --CHO R¹³ is H, R is C(═O)R⁴.
 6. A compound ofclaim 1 whereinR¹² is --CHO R¹³ is H, R is C(R²)OR⁵.
 7. A compound ofclaim 1 whereinR¹² is --CHO R¹³ is H, R is C(R⁶)(R⁷)(R⁸).
 8. A compoundof claim 1 whereinR¹² is --CHO R¹³ is H; R is ##STR42##
 9. A compound ofclaim 1 wherein R¹² is --CHO,R¹³ is H, R is CH(R⁶)(R⁷), R⁶ is(a) H, (b)C₁ -C₁₄ alkyl, (c) aryl, (d) aryl C₁₋₆ alkyl, (e) --(CH₂)_(y) CHR¹¹(CH₂)_(z) H, (f) --(CH₂)_(y) C(R⁷)═CH(CH₂)_(z) H, R⁷ is H or C₁ -C₆alkyl, R¹¹ is OH.
 10. A compound of claim 1 whereinR¹² is --CHO R¹³ isH, R is(a) --CH₃, (b) --CH₂ CH₃, (c) --CH₂ CH₂ CH₃, (d) --CH₂ CH₂ CH₂CH₃, (e) --CH₂ CH₂ CH₂ CH₂ CH₃, (f) --CH₂ CH₂ CH₂ CH₂ CH₂ CH₃, (g) CH₂CH₂ CH₂ CH₂ CH₂ CH₂ CH₃, (h) --CH(CH₃)₂, (i) --CH₂ CH(CH₃)₂, (j) --CH₂CH₂ CH(CH₃)₂, (k) --CH₂ C₆ H₅, (l) --CH₂ CH═CHCH₃, (m) --CH₂ CH═CHCH₂CH₃, (n) --CH₂ CH═CHCH₂ CH₂ CH₃, (o) --CH₂ CH═CHCH₂ CH₂ CH₂ CH₃.
 11. Acompound of claim 1 whereinR¹² is --CHO R¹³ is(a) H, R is(a) --CH₂ CH₂CH₃, (b) --CH₂ CH₂ CH₂ CH₃, (c) --CH₂ CH₂ CH₂ CH₂ CH₃, (d) --CH₂CH(CH₃)₂, (e) --CH₂ CH₂ CH(CH₃)₂, (f) (Z)--CH₂ CH═CHCH₂ CH₃, (g)(Z)--CH₂ CH═CHCH₂ CH₂ CH₃.
 12. A pharmaceutical composition whichcomprises a compound of claim 1 and a pharmaceutically acceptablecarrier.
 13. An agrochemical composition which comprises a compound ofclaim 1 and a agriculturally acceptable carrier.
 14. A method for thetreatment or prevention of fungal infection in an animal which comprisesadminstering to said animal an antifungal effective amount of a compoundof claim
 1. 15. A method for controlling phytopathogenic fungi whichcomprises administering to a plant in need of such control an antifungaleffective amount of a compound of claim 1.